Hematopoietic Cell E-Selectin/L-Selectin Ligand Glycosylated CD44 Polypeptide

ABSTRACT

The invention feature methods and compositions for treating hematopoietic disorders, inflammatory conditions, and cancer and providing stem cell therapy in a mammal.

RELATED APPLICATIONS

This application claims priority to U.S. Ser. No. 60/240,987, filed,Oct. 18, 2000 and U.S. Ser. No. 60/297,474, filed, Jun. 11, 2001 whichare incorporated herein by reference in their entireties.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with U.S. government support under NationalInstitutes of Health grants NHLBI RO1 HL60528 and CA84156. Thegovernment has certain rights in the ID invention.

FIELD OF THE INVENTION

The invention provides compositions and methods for identifying stemcells and treating hematopoietic disorders, (e.g., leukemia), cancer,inflammatory disorders and disorders amenable for treatment with stemcells, (e.g., myocardial infarction, Parkinson's disease, diabetes, orstroke).

BACKGROUND OF THE INVENTION

The specialized cytoarchitecture of the hematopoietic microenvironmentis created by discrete cell-cell and cell-matrix adhesive interactionsthat are tightly regulated by lineage-specific expression of adhesionmolecules. The earliest human hematopoietic progenitor cells (HPCs) arecharacterized by the absence of lineage-specific markers and expressionof the cell surface molecule, CD34. A variety of adhesion molecules areexpressed on HPCs, including CD44 (the “hyaluronic acid receptor”, alsoknown as H-CAM), members of the integrin (e.g., LFA-1, VLA-4) andimmunoglobulin (e.g., ICAM1) superfamilies, and a member of the selectinfamily, L-selectin. L-selectin (CD62L) is a calcium-dependent,carbohydrate binding protein in a family of adhesion molecules that alsoincludes E-selectin (CD62E), expressed on activated vascularendothelium, and P-selectin (CD62P), found on both activated plateletsand endothelial cells. Selectin-mediated interactions are critical notonly for the rapid and efficient recruitment of leukocytes at a site ofinjury, but for steady state, tissue-specific homing as illustrated in:(1) lymphocyte homing to peripheral lymph nodes, (2) cutaneous tropismof human skin-homing T-cells and (3) hematopoietic progenitor cell (HPC)entry into bone marrow.

SUMMARY OF THE INVENTION

The invention features a novel glycosylated polypeptide expressed onnormal human hemopoietic progentitor cells and on leukemic blastsdesignated hematopoietic cell E-selection/L-selectin ligand (HCELL). TheHCELL polypeptides are also referred to herein as “KG1a CD44”. HCELL isa novel glycoform of CD44 containing HECA-452 reactive sialyated,fucosylated N-glycans. The HCELL polypeptide is a ligand for bothL-selectin and E-selectin.

The invention also features a method of identifying stem cells bycontacting a test cell population with one or more agents thatspecifically bind to HCELL under conditions sufficient to form a complexbetween the agent and stem cell. The complex is detected and if presentindicates the cell is a stem cell. Suitable agents include an anti-CD44antibody, or an antibody with the binding specificity of monoclonalantibody HECA-452.

A stem cell is also identified by providing a selectin polypeptide,e.g., E-selectin or L-selectine immobilized on a solid phase andcontacting the solid phase with a fluid sample containing a suspensionof test cells. The solid phase is contacted with the fluid sample shearstress is achieved at the surface of the solid phase. By observing whichcells that adhere to the solid phase a stem cell is identified. The testcell can be, for example, blood or bone marrow.

Also provided by the invention are various methods of isolating a stemcell (e.g., a pluripotent stem cell) from a population of cells. A stemcell is isolated by contacting a cell population with one or more agentsthat specifically bind to a HCELL polypeptide under conditionssufficient to form a complex between the agents and a stem cell. Complexformation is detected and removed from the cell population therebyisolating the stem cell from the cell population. Additionally, the stemcell/agent complex is disrupted.

Alternatively, a stem cell is isolated by providing a selectinpolypeptide, e.g., E-selectin or L-selection immobilized on a solidphase and contacting the solid phase with a fluid sample containing asuspension of cells. The solid phase is contacted with the fluid sampleso that shear stress is achieved at the surface of the solid phase. Stemcells are isolated by recovering the cells that adhere to the solidphase.

The invention also features methods of treating a hematopoieticdisorders, cancer and disorders amenabke to treatment with stem cells(i.e., stem cell therapy) in a mammal, by comprising administering tothe mammal a composition comprising the cells isolated according to themethods described above.

The invention also provides a method of increasing the affinity of acell for E-selectin and/or L-selectin, by providing a cell andcontacting the cell with one or more agents that increases cell-surfaceexpression or activity a HCELL polypeptide, thereby increasing affinityof the cell for E-selectin and/or L-selectin. Suitable agents includefor example, a nucleic acid that encodes a CD44, glycosyltransferase ora glycosidase polypeptide.

The invention features methods of increasing the engraftment potentialof a stem cell, by providing a stem cell a and contacting the stem cellwith one or more agents that increases cell-surface expression oractivity a HCELL polypeptide on the cell, thereby increasing theengraftment potential of stem cell.

Alternatively, the engraftment potential of a cell population isincreased by providing a selectin polypeptide, e.g. E-selectin orL-selectin immobilized on a solid phase and contacting the solid phasewith a fluid sample containing a cell population. The solid phase iscontacted with the fluid sample shear stress is achieved at the surfaceof the solid phase. Cells that adhere to the solid phase are recovered.

Levels of engrafted stem cells in a subject, e.g., human are increasedby administering to the subject an agent that increases cell-surface orexpression of a HCELL polypeptide in the subject. Suitable agentsinclude for example, a nucleic acid that encodes a CD44,glycosyltransferase or a glycosidase polypeptide. Alternatively, levelsof engrafted stem cells in a subject are increased by administering tothe subject a composition containing the cells isolated according to theabove described methods.

The invention also features methods of treating hematopoietic disorders,e.g., leukemia in a subject. The hematopoietic disorder is treated byadministering to the subject an agent that decreases the cell-surface orexpression of a HCELL polypeptide in the subject. Alternatively, thehematopoietic disorder is treated by providing blood from the subjectand contacting the blood with one or more agents that specifically bindto a HCELL polypeptide under conditions sufficient to form a complexbetween the agents and a blood cell. The complex is detected, if presentand removed from the blood. The blood is re-introduced to the subject.

Additionally, a hematopoietic disorder is treated by providing bloodfrom the subject and a selectin polypeptide, e.g., E-selectin orL-selectin immobilized on a solid phase and contacting the solid phasewith a the blood. The solid phase is contacted with the blood sample soshear stress is achieved at the surface of the solid phase. The blood isthen re-introduced onto the subject.

Further the hematopoietic disorder is treated by administering to thesubject an agent that specifically bind to the HCELL glycoprotein.

The invention also features, a method of treating an inflammatorydisorder in a subject, by administering to a subject a HCELLgylcoprotein or fragment thereof.

The invention further features a method of diagnosing or determining thesusceptibility to a hematologic disorder in a subject, by contacting asubject derived cell population with one or more agents thatspecifically bind a HCELL glycprotein under conditions sufficient toform a complex between the agent and cell, and detecting the complex.The presence of the complex indicates the presence of or thesusceptibility to a hematologic disorder in the subject

Additionally, the invention feature a method of determining theprognosis or efficacy of treatment of a hematologic disorder in asubject, by contacting a subject derived cell population with one ormore agents that specifically bind a HCELL gycoproetin under conditionssufficient to form a complex between the agent and cell, if anddetecting the complex.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a photograph of a Western Blot showing expressionHECA-452-reactive epitopes on various human hematopoietic cell lines.

FIG. 1B is a bar graph showing cell tethering and rolling ofhematopoietic cell lines (shear stress of 2.8 dynes/em2) onglutaraldehyde-fixed monolayers. Data are presented as mean±S.D. CHO-Ecell rolling per field ×5 fields, minimum of three experiments.

FIG. 2 is a bar graph showing human hematopoietic cell rolling onfreshly isolated human bone marrow endothelial cells.

FIG. 3A is bar chart showing the results of a blot rolling assay ofE-selectin ligand activity.

FIG. 3B is a photograph of a Western Blot showing the effect ofN-glycosidase-F treatment on HECA-452 staining.

FIG. 3C is photograph of a Western Blot showing PSGL-1 expression onKG1a cells.

FIG. 3D is a photograph of a membrane of a Blot Rolling Assay showingthe effect of N-glycosidase-F on E-selection ligand activity.

FIG. 4A is a photograph of a membrane of a Blot Rolling Assay showingthe E-selectin binding activity of HCELL exhaustively immunoprecipitatedwith anti-CD44 antibody (Hermes-1). (Pre-Ippt): Total KGia lysate 10 ug;(lane 1): First round Hermes-1 immunoprecipitate; (lane 2): Second roundHermes-1 immunoprecipitate; (lane 3): Third round Hermes-1immunoprecipitate; (lane 4): Residual lysate after three rounds ofHermes-1 immunoprecipitation.

FIG. 4 B is a photograph of a membrane of a Blot Rolling Assay showingthe E-selectin binding activity of exhaustively immunoprecipitated HCELLwith Hermes-1 antibody and blot stained with HECA-452 antibody.(Pre-Ippt): Total KGia lysate 10 ug; (lane 1): First round Hermes-1immunoprecipitate; (lane 2): Second round Hermes-1 immunoprecipitate;(lane 3): Third round Hermes-1 immunoprecipitate; (lane 4): Residuallysate after three rounds of Hermes-1 immunoprecipitation.

FIG. 5A is a bar chart of E-selectin-mediated CHO-E cell rolling.Rolling was observed at 2.8 dynes/cm2 on KG1a CD44, but wassignificantly lower on KG1a PSGL-1 at 2.8 dynes/cm2 (p<0.001).N-glycosidase-F- and a-L-fucosidase-treated KG1a CD44, and Vibriocholerae neuraminidase treatment of KG1a membrane protein abrogatedCHO-E cell rolling (p<0.001)

FIG. 5B is a bar chart showing CHO-P cell rolling. Rolling was observedon KG1a PSGL-1 but not on KG1a CD44 (2.8 dynes/cm2). No rolling wasobserved on negative controls (CHO-Mock cells and CHO-P cells pretreatedwith function blocking anti-P-selectin moAb AK-4 (10 ug/ml)).

FIG. 6A is a photograph of a membrane of a Blot Rolling Assay showingHCELL activity of various hematopoietic cells. (1) Human BM mononuclearcells 10⁸ cells), (2) CD34−/lineage+ cells (10⁷ cells), (3)CD34+/lineage− cells (10⁷ cells), (4) CD34−/lineage+ cells (10⁸ cells).

FIG. 6B is a photograph of a Western Blot illustrating the effect ofN-glycosidase-F treatment on HECA-452 activity on circulating blastsfrom adult myelogenous leukemia (AML).

FIG. 6C is a photograph of a membrane showing blot rolling assay resultsof AML (M5) membrane protein (50 ug) immunoprecipitated with isotypecontrol or with Hermes-1 moAb, and of N-glycosidase-F treated Hermes-1immunoprecipitates.

FIG. 6D is a photograph showing HECA-452 staining of immunoprecipitatedCD44 from membrane preparations (50 ug) of an AML (M0), AML (M1) andatypical CML (brc/abl), and of human BM endothelial cell line (BMEC-1,100 ug total protein) that were evaluated for E-selectin ligandactivity. E-selectin ligand activity correlates with intensity ofHECA-452 staining of 100 kDa band.

FIG. 6E is photograph of a Western Blot showing CD44 expression ofBMEC-1.

FIG. 7A is a photograph of a Western blot of HECA-452-reactive membraneproteins (30 μg/lane) from human leukemia cell lines K562 (lane 1),RPMI-8402 (lane 2), HL-60 (lane 3), KG1a (lane 4), neuraminidase-treatedKG1a (lane 5), and OSGE-treated KG1a (lane 6).

FIG. 7B is a photograph of a Western blot showing the re-expression ofHECA-452-reactive proteins on KG1a whole cells after neuraminidase andthen tunicamycin treatment. Lanes: 1, untreated; 2, neuraminidase; 3,neuraminidase then 24-h DMSO recovery; and 4, neuraminidase then 24-htunicamycin recovery.

FIG. 7C is a photograph of an autoradiograph of KG1a membrane proteinsmetabolically radiolabeled with 2-[³H]-mannose resolved on reducing 6%SDS-PAGE.

FIG. 7D is a bar chart showing L-selectin-dependent, lymphocytetethering and rolling on blotting membrane under hydrodynamic flowconditions (2.3 dynes/cm²) over the 98, 120 and 130 kDa HECA-452-bearingKG1a proteins.

FIG. 8 is a photograph of a Western Blot showing the 98 kDa gel fragmentfrom the third step of the 3-Step SDS-PAGE (6 to 9% bis/acrylamide)purification schema reactivity with either HECA-452 (0.4 μg/ml) oranti-CD44 moAbs (A3D8 or Hermes-1 (1 μg/ml)).

FIG. 9 is a photograph of a membrane showing L-selectin mediated blotrolling assay results of immunoprecipitated KG1a CD44 immunostained witheither Hermes-1 (left) or HECA-452 (right).

FIG. 10A is a photograph of a membrane showing blot rolling assayresults of treatment with N-glycosidase-F on L-selectin ligand activityof immunoprecipitated CD44 from KG1a cells.

FIG. 10B is a photograph of a membrane showing blot rolling assayresults of treatment with N-glycosidase-F on L-selectin ligand activityof immunoprecipitated CD44 from AML (M5) blasts.

FIG. 11A is a photograph of an autoradiogram of immunoprecipitated CD44from non-chlorate-and chlorate-treated KG1a cells radiolabeled with[³⁵S]SO₄

FIG. 11B is a photograph of HECA-452 immunoblots of hnmunoprecipitatedKG1a CD44 from chlorate (+) and non-chlorate treated (−)KG1a cells.L-selectin ligand activity (mean number of lymphocytes/200× mag. field/5fields) was equivalent in sulfated and non-sulfated (chlorate treated)KG1a CD44.

FIG. 12A is a bar chart showing the results of lymphocyte rolling onglutaraldehyde-fixed hematopoietic cell lines, KG1a, HL60, K562 and RPMI8402 over a range of shear stress.

FIG. 12B is a bar chart showing the results of neutrophil rolling onglutaraldehyde-fixed hematopoietic cell lines, KG1a, HL60, K562 and RPMI8402 over a range of shear stress.

FIG. 12 C is graph showing the results of the shear-basedStamper-Woodruff assay evaluating the ability of KG1a, HL60, K562 andRPMI 8402 cell lines to support L-selectin-mediated lymphocyte bindingover a range of rpms. Mean lymphocyte adherence to KG1a cells was10-fold greater than on HL60 cells (Student's paired t-test; p<0.001).All L-selectin-mediated lymphocyte adherence was prevented bypretreating lymphocytes with anti-L-selectin monoclonal antibodies (10μg/ml), by using PMA-treated lymphocytes, and or by using assay mediumcontaining 5 mM EDTA.

FIG. 13A is a bar chart showing rolling of CHO-P-selectin or mocktransfectants on glutaraldehyde-fixed hematopoietic cell monolayers wasmeasured in the parallel-plate flow chamber. KG1a and HL60 cellssupported equivalent P-selectin-mediated CHO-P cell rolling at 0.4dynes/cm².

FIG. 13B is a chart showing CHO-P cell rolling interactions on KG1a andHL60 cells that were measured over a similar shear stress range and wereeliminated in the presence of EDTA and prevented by pretreating KG1a andHL60 cells with mocarhagin (10 μg/ml).

FIG. 14A is a photograph of an autoradiograph of Hermes-1 (CD44) andPL-2 (PSGL-1) immunoprecipitates and L-selectin ligand activity of HCELLand PSGL-1.

FIG. 14B is a chart showing the results of a Stamper-Woodruff assays ofimmunoprecipitated KG1a CD44 (1.5 μg) and PSGL-1 (3 μg).

FIG. 15 A-F are photomicrographs of lymphocytes bound to human HC CD44or PSGL-1 in the Stamper-Woodruff Assay. Immunoaffinity purified KG1a orAML (M1) CD44 (1.5 μg) and KG1a PSGL-1 (2 or 6 μg) were prepared asdescribed in the Methods and analyzed for L-selectin-mediated lymphocyteadherence in the Stamper-Woodruff assay. KG1a or AML (M1) CD44 (Panels Aand D, respectively) supported a distinctively higher number oflymphocytes than to respective N-glycosidase F-treated CD44 (Panels Band E) and to isotype control immunoprecipitates, monoclonal Ab(Hermes-1) alone or L-selectin immunoprecipitate (All represented byPanels C and F for KG1a and AML (M1), respectively). Anti-L-selectin Abs(10 μg/ml), 5 mM EDTA-containing assay medium or PMA-treated (50 ng/ml)lymphocytes completely inhibited lymphocyte adherence (also indicated inPanels C and F from KG1a and AML (M1), respectively) verifying thespecificity of lymphocyte L-selectin in this assay system.

FIG. 16 is a photograph of a Western Blot showing HECA-452 epitope(s) onKG1a CD44 and PSGL-1.

FIG. 17A-B are photographs showing the expression ofglycosyltransferases (FucTIV, FucTVII and ST3GalIV) on humanhematopoietic cell Lines. (Panel A) RT-PCR expression analysis of FucTIVand FucTVII. Lane 1: FucTIV, Lane 2: FucTVII, Lane 3: β-actin, and Lane4: ddH₂O. (Panel B) RT-PCR expression analysis of ST3GalIV. Lane 1:ST3GalIV, Lane 2: β-actin, and Lane 3: ddH₂O.

DETAILED DESCRIPTION

The present invention is based in part on the discovery of a novelglycosylated polypeptide expressed on normal human hemopoeticprogentitor cells and on leukemic blasts designated hematopoietic cellE-selection/L-selectin ligand (HCELL). The HCELL polypeptides are alsoreferred to herein as “KG1a CD44”. Using blot rolling assay it wasdemonstrated that HCELL is novel glycoform of CD44 containing HECA-452reactive sialyated, fucosylated N-glycans. The HCELL polypeptide is aligand for both a L-selectin and E-selectin. HCELL L-selectin andE-selectin ligand activity requires sialofucosylated N-linked glycansthat are recognized by rat monoclonal antibody HECA-452 and issulfation-independent.

CD44 is a broadly distributed cell surface glycoprotein receptor for theglycosamino glycan hyaluronan (HA) which is a major component ofextracellular spaces. It is expressed on a diverse variety of cell typesincluding most hematopoietic cells, keratinocytes, chondrocytes, manyepithelial cell types, and some endothelial and neural cells. CD44 isknown to participate in a wide variety of cellular functions, includingcell-cell aggregation, retention of pericellular matrix, matrix-cell andcell-matrix signaling, receptor-mediated internalization/degradation ofhyaluronan, and cell migration. All these functions are dependent uponCD44-hyaluronan interactions and is sulfation dependent.

The gene encoding CD44 gene consists of 20 exons (19 exons in earlierliterature, exons 6a and 6b have been reclassified as exons 6 and 7, tomake 20 exons total). Although a single gene located on the short arm ofhuman chromosome 11 encodes CD44, multiple mRNA transcripts that arisefrom the alternative splicing of 12 of the 20 exons have beenidentified. The standard and most prevalent form of CD44 (termed CD44s)consists of a protein encoded by exons 1-5, 16-18, and 20. This form isthe most predominant form on hematopoetic cells, and is also known asCD44H. CD44s exhibits the extracellular domains (exons 1-5 and 16), thehighly conserved transmembrane domain (exon 18), and the cytoplasmicdomain (exon 20). The 1482 by of open reading frame mRNA for human CD44sresults in translation of a polypeptide chain of ˜37 kDa.Post-translational addition of N-linked and O-linked oligosaccharidescontribute to the ˜85-kDa molecular mass of the final CD44 protein asestimated by SDS-PAGE.

The standard or hematopoietic isoform of CD44 (CD44H) is a type 1transmembrane molecule consisting of ˜270 amino acids (aa) ofextracellular domain (including 20 aa of leader sequence, a 21 aatransmembrane domain and a 72 aa cytoplasmic domain. The amino terminal˜180 aa are conserved among mammalian species (˜85% homology). Thisregion contains six conserved cysteines, and six conserved consensussites for N glycosylation. Five conserved consensus sites forN-glycosylation are located in the amino terminal 120 aa of CD44. Allfive sites appear to be utilized in the murine and human cell lines.Several studies have shown that cell specific N-glycosylation canmodulate the HA binding function of CD44. Cell lines and normal B-cellsshowed differenced in N-glycosylation associated with different HAbinding states. In particular, CD44 from HA binding cells had lessglycosylation than from non-HA binding cells. Additionally, removal ofsialic acids (both from the cell surface and from CD44-Ig fusionproteins) enhances HA binding. Thus the HCELL CD44 glycoform of theinvention is unlike any previously described CD44.

In contrast the non-conserved region (˜aa 183 to 256) shows only ˜35%similarity between mammalian species. This region contains potentialsites for numerous carbohydrate modifications of CD44 and the site ofalternative splicing which allows for the insertion of extra amino acidsequence from variable exons of the CD44 gene.

A HCELL polypeptide comprises an amino acid sequence of CD44 and bindsto antibody having the binding specificity of monoclonal antibodyHECA-452. (ATCC Number: HB-11485) HECA-452 recognizes cutaneouslymphocyte associated antigen. HECA-452 binding of HCELL decrease afterN-glycosidase-F, sialidase or fucosidase treatment. Furthermore, HCELLactivity, e.g., E-selectin and L-selectin binding also decreases uponN-glycosidase-F, sialidase, or fucosidase treatment demonstrating theimportance of the sialofucosylated N-linked glycans in HCELL function.In contrast, sialylation of CD44 inhibits binding of CD44 to hyaluronicacid. Moreover, CD44 binding to hyaluronate is increased by sulfation,but sulfation is not necessary for the E- and L-selectin activity ofHCELL.

Preferably the CD44 polypeptide is the standard or hematopoietic isoformof CD44 (CD44H). Alternately, the CD44 polypeptide is the R1 (CD44R1) orR2 isoform (CD44R2). For example, a HCELL polypeptide comprises theamino acid sequence of SEQ ID NO:1. (Gen Bank Acc. CAA40133; Table 1) AHCELL polypeptide is at least about 30%, 50%, 70%, 80%, or 95% identicalto the polypeptide sequence of SEQ ID NO:1.

TABLE 1 1mdkfwwhaaw glclvplsla qidlnitcrf agvfhvekng rysisrteaa dlckafnstl 61 ptmaqmekal sigfetcryg fieghvvipr ihpnsicaan ntgvyiltsn tsqydtycfn 121asappeedct svtdlpnafd gpititivnr dgtryvqkge yrtnpediyp snptdddvss 181gssserssts ggyifytfst vhpipdedsp witdstdrip atnmdsshst tlqptanpnt 241glvedldrtg plsmttqqsn sqsfstsheg leedkdhptt stltssnrnd vtggrrdpnh 301segsttlleg ytshyphtke srtfipvtsa ktgsfgvtav tvgdsnsnvn rslsgdqdtf 361hpsggshtth gsesdghshg sqeggantts gpirtpqipe wliilaslla lalilavcia 421vnsrrrcgqk kklvinsgng avedrkpsgl ngeasksqem vhlvnkesse tpdqfmtade 481trnlqnvdmk igv (SEQ ID NO: 1)

HCELL Polypeptides

One aspect of the invention pertains to isolated HCELL glycoproteins,and biologically active portions thereof, or derivatives, fragments,analogs or homologs thereof. Also provided are polypeptide fragmentssuitable for use as immunogens to raise anti-HCELL antibodies. In oneembodiment, native HCELL glycoproteins can be isolated from cells ortissue sources by an appropriate purification scheme using standardprotein purification techniques.

In another embodiment, HCELL glycoproteins are produced by recombinantDNA techniques. Alternative to recombinant expression, a HCELLglycoprotein or polypeptide can be synthesized chemically using standardpeptide synthesis techniques.

An “isolated” or “purified” protein or biologically active portionthereof is substantially free of cellular material or othercontaminating proteins from the cell or tissue source from which theHCELL glycoprotein is derived, or substantially free from chemicalprecursors or other chemicals when chemically synthesized. The language“substantially free of cellular material” includes preparations of HCELLglycoprotein in which the protein is separated from cellular componentsof the cells from which it is isolated or recombinantly produced. In oneembodiment, the language “substantially free of cellular material”includes preparations of HCELL glycoprotein having less than about 30%(by dry weight) of non-HCELL glycoprotein (also referred to herein as a“contaminating protein”), more preferably less than about 20% ofnon-HCELL glycoprotein, still more preferably less than about 10% ofnon-HCELL glycoprotein, and most preferably less than about 5% non-HCELLglycoprotein. When the HCELL glycoprotein or biologically active portionthereof is recombinantly produced, it is also preferably substantiallyfree of culture medium, i.e., culture medium represents less than about20%, more preferably less than about 10%, and most preferably less thanabout 5% of the volume of the protein preparation.

The language “substantially free of chemical precursors or otherchemicals” includes preparations of HCELL glycoprotein in which theprotein is separated from chemical precursors or other chemicals thatare involved in the synthesis of the protein. In one embodiment, thelanguage “substantially free of chemical precursors or other chemicals”includes preparations of HCELL glycoprotein having less than about 30%(by dry weight) of chemical precursors or non-HCELL chemicals, morepreferably less than about 20% chemical precursors or non-HCELLchemicals, still more preferably less than about 10% chemical precursorsor non-HCELL chemicals, and most preferably less than about 5% chemicalprecursors or non-HCELL chemicals.

Biologically active portions of a HCELL glycoprotein include peptidescomprising amino acid sequences sufficiently homologous to or derivedfrom the amino acid sequence of the HCELL glycoprotein, e.g., the aminoacid sequence shown in SEQ ID NO: 1, that include fewer amino acids thanthe full length HCELL glycoproteins, and exhibit at least one activityof a HCELL glycoprotein, e.g., HECA-452 antibody reactivity, anti-CD44antibody reactivity. E-selectin binding, or L-selectin binding.Typically, biologically active portions comprise a domain or motif withat least one activity of the HCELL glycoprotein, e.g., N-linkedglycosylation sites. A biologically active portion of a HCELLglycoprotein can be a polypeptide which is, for example, 10, 25, 50, 100or more amino acids in length. Preferably the biologically activeportion of a HCELL glycoprotein includes the amino acid sequence of theN-terminal domain of a CD44 polypeptide.

The invention may contain at least one of the above-identifiedstructural domains. Moreover, other biologically active portions, inwhich other regions of the protein are deleted, can be prepared byrecombinant techniques and evaluated for one or more of the functionalactivities of a native HCELL glycoprotein, e.g., E-selectin, L-selectionbinding activity. In an embodiment, the HCELL glycoprotein has an aminoacid sequence shown in SEQ ID NO: 1. In other embodiments, the HCELLglycoprotein is substantially homologous to SEQ ID NO: 1 and retains thefunctional activity of the protein of SEQ ID NO: 1a yet differs in aminoacid sequence due to natural allelic variation or mutagenesis, asdescribed in detail below. Accordingly, in another embodiment, the HCELLglycoprotein is a protein that comprises an amino acid sequence at leastabout 45% homologous to the amino acid sequence of SEQ ID NO: 1 andretains the functional activity of the HCELL glycoproteins of SEQ IDNO: 1. Alternatively, a HCELL polypeptide has a CD44 amino acid sequencecapable of N-linked glycosylation.

Chimeric and Fusion Proteins

The invention also provides HCELL chimeric or fusion proteins. As usedherein, a HCELL “chimeric protein” or “fusion protein” comprises a HCELLpolypeptide operatively linked to a non-HCELL polypeptide. A “HCELLpolypeptide” refers to a polypeptide having an amino acid sequencecorresponding to HCELL, whereas a “non-HCELL polypeptide” refers to apolypeptide having an amino acid sequence corresponding to a proteinthat is not substantially homologous to the HCELL glycoprotein, e.g., aprotein that is different from the HCELL glycoprotein and that isderived from the same or a different organism. Within a HCELL fusionprotein the HCELL polypeptide can correspond to all or a portion of aHCELL glycoprotein. In one embodiment, a HCELL fusion protein comprisesat least one biologically active portion of a HCELL glycoprotein. Inanother embodiment, a HCELL fusion protein comprises at least twobiologically active portions of a HCELL glycoprotein. In yet anotherembodiment, a HCELL fusion protein comprises at least three biologicallyactive portions of a HCELL glycoprotein. Within the fusion protein, theterm “operatively linked” is intended to indicate that the HCELLpolypeptide and the non-HCELL polypeptide are fused in-frame to eachother. The non-HCELL polypeptide can be fused to the N-terminus orC-terminus of the HCELL polypeptide. For example, in one embodiment aHCELL fusion protein comprises a HCELL anti-CD44 binding domain operablylinked to the extracellular domain of a second protein. Such fusionproteins can be further utilized in screening assays for compounds whichmodulate HCELL activity.

In one embodiment, the fusion protein is a GST-HCELL fusion protein inwhich the HCELL sequences are fused to the C-terminus of the GST (i.e.,glutathione S-transferase) sequences. Such fusion proteins canfacilitate the purification of recombinant HCELL. In another embodiment,the fusion protein is a HCELL glycoprotein containing a heterologoussignal sequence at its N-terminus. For example, the native HCELL signalsequence can be removed and replaced with a signal sequence from anotherprotein. In certain host cells (e.g., mammalian host cells), expressionand/or secretion of HCELL can be increased through use of a heterologoussignal sequence.

In one embodiment, the fusion protein is a HCELL-immunoglobulin fusionprotein in which the HCELL sequence of fragement thereof are fused tosequences derived from a member of the immunoglobulin protein family.The HCELL-immunoglobulin fusion proteins of the invention can beincorporated into pharmaceutical compositions and administered to asubject to inhibit an interaction between a HCELL ligand and a HCELLglycoprotein on the surface of a cell, to thereby suppressHCELL-mediated signal transduction in vivo. The HCELL-immunoglobulinfusion proteins can be used to affect the bioavailability of a HCELLcognate ligand. Inhibition of the HCELL ligand/HCELL interaction may beuseful therapeutically for both the treatment of proliferative anddifferentiative disorders, as well as modulating (e.g. promoting orinhibiting) cell survival.

Moreover, the HCELL-immunoglobulin fusion proteins of the invention canbe used as immunogens to produce anti-HCELL antibodies in a subject, topurify HCELL ligands, and in screening assays to identify molecules thatinhibit the interaction of HCELL with a HCELL ligand.

A HCELL chimeric or fusion protein of the invention can be produced bystandard recombinant DNA techniques. For example, DNA fragments codingfor the different polypeptide sequences are ligated together in-frame inaccordance with conventional techniques, e.g., by employing blunt-endedor stagger-ended termini for ligation, restriction enzyme digestion toprovide for appropriate termini, filling-in of cohesive ends asappropriate, alkaline phosphatase treatment to avoid undesirablejoining, and enzymatic ligation. In another embodiment, the fusion genecan be synthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of gene fragments can becarried out using anchor primers that give rise to complementaryoverhangs between two consecutive gene fragments that can subsequentlybe annealed and reamplified to generate a chimeric gene sequence (see,for example, Ausubel et al. (eds.) CURRENT PROTOCOLS IN MOLECULARBIOLOGY, John Wiley & Sons, 1992). Moreover, many expression vectors arecommercially available that already encode a fiision moiety (e.g., a GSTpolypeptide). A HCELL-encoding nucleic acid can be cloned into such anexpression vector such that the fusion moiety is linked in-frame to theHCELL glycoprotein.

HCELL Agonists and Antagonists

The present invention also pertains to variants of the HCELLglycoproteins that function as either HCELL agonists (mimetics) or asHCELL antagonists. Variants of the HCELL glycoprotein can be generatedby mutagenesis, e.g., discrete point mutation or truncation of the HCELLglycoprotein. An agonist of the HCELL glycoprotein can retainsubstantially the same, or a subset of, the biological activities of thenaturally occurring form of the HCELL glycoprotein. An antagonist of theHCELL glycoprotein can inhibit one or more of the activities of thenaturally occurring form of the HCELL glycoprotein by, for example,competitively binding to a downstream or upstream member of a cellularsignaling cascade which includes the HCELL glycoprotein. Thus, specificbiological effects can be elicited by treatment with a variant oflimited function. In one embodiment, treatment of a subject with avariant having a subset of the biological activities of the naturallyoccurring form of the protein has fewer side effects in a subjectrelative to treatment with the naturally occurring form of the HCELLglycoproteins.

Variants of the HCELL glycoprotein that function as either HCELLagonists (mimetics) or as HCELL antagonists can be identified byscreening combinatorial libraries of mutants, e.g., truncation mutants,of the HCELL glycoprotein for HCELL glycoprotein agonist or antagonistactivity. In one embodiment, a variegated library of HCELL variants isgenerated by combinatorial mutagenesis at the nucleic acid level and isencoded by a variegated gene library. A variegated library of HCELLvariants can be produced by, for example, enzymatically ligating amixture of synthetic oligonucleotides into gene sequences such that adegenerate set of potential HCELL sequences is expressible as individualpolypeptides, or alternatively, as a set of larger fusion proteins(e.g., for phage display) containing the set of HCELL sequences therein.There are a variety of methods which can be used to produce libraries ofpotential HCELL variants from a degenerate oligonucleotide sequence.Chemical synthesis of a degenerate gene sequence can be performed in anautomatic DNA synthesizer, and the synthetic gene then ligated into anappropriate expression vector. Use of a degenerate set of genes allowsfor the provision, in one mixture, of all of the sequences encoding thedesired set of potential HCELL sequences. Methods for synthesizingdegenerate oligonucleotides are known in the art (see, e.g., Narang(1983) Tetrahedron 39:3; Itakura et al. (1984) Annu Rev Biochem 53:323;Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucl Acid Res11:477.

Anti-HCELL Antibodies

The invention encompasses antibodies and antibody fragments, such asF_(ab) or (F_(ab))₂, that bind immunospecifically to any of thepolypeptides of the invention. An isolated HCELL glycoprotein, or aportion or fragment thereof, can be used as an immunogen to generateantibodies that bind HCELL using standard techniques for polyclonal andmonoclonal antibody preparation. The full-length HCELL glycoprotein canbe used or, alternatively, the invention provides antigenic peptidefragments of HCELL for use as immunogens. The antigenic peptide of HCELLcomprises at least 4 amino acid residues of the amino acid sequenceshown in SEQ ID NO: 1 and encompasses an epitope of HCELL such that anantibody raised against the peptide forms a specific immune complex withHCELL. Preferably, the antigenic peptide comprises at least 6, 8, 10,15, 20, or 30 amino acid residues. Longer antigenic peptides aresometimes preferable over shorter antigenic peptides, depending on useand according to methods well known to someone skilled in the art. Morepreferably the antigenic peptide comprises at least one N-linkedglycosylation site.

In certain embodiments of the invention, at least one epitopeencompassed by the antigenic peptide is a region of HCELL that islocated on the surface of the protein, e.g., a hydrophilic region. As ameans for targeting antibody production, hydropathy plots showingregions of hydrophilicity and hydrophobicity may be generated by anymethod well known in the art, including, for example, the Kyte Doolittleor the Hopp Woods methods, either with or without Fouriertransformation. See, e.g., Hopp and Woods, 1981, Proc. Nat. Acad ScL USA78: 3824-3828; Kyte and Doolittle 1982, J. Mol. Biol. 157: 105-142, eachincorporated herein by j: reference in their entirety.

As disclosed herein, HCELL glycoprotein sequence, or derivatives,fragments, analogs or homologs thereof, may be utilized as immunogens inthe generation of antibodies that immunospecifically-bind these proteincomponents. The term “antibody” as used herein refers to immunoglobulinmolecules and immunologically active portions of immunoglobulinmolecules, i.e., molecules that contain an antigen binding site thatspecifically binds (immunoreacts with) an antigen, such as HCELL. Suchantibodies include, but are not limited to, polyclonal, monoclonal,chimeric, single chain, F_(ab) and F_((ab′)2) fragments, and an F_(ab)expression library. In a specific embodiment, antibodies to human HCELLglycoproteins are disclosed. Various procedures known within the art maybe used for the production of polyclonal or monoclonal antibodies to aHCELL glycoprotein sequence of SEQ ID NO: 1, or derivative, fragment,analog or homolog thereof. Some of these proteins are discussed below.

For the production of polyclonal antibodies, various suitable hostanimals (e.g., rabbit, goat, mouse or other mammal) may be immunized byinjection with the native protein, or a synthetic variant thereof, or aderivative of the foregoing. An appropriate immunogenic preparation cancontain, for example, recombinantly expressed HCELL glycoprotein or achemically synthesized HCELL polypeptide. The preparation can furtherinclude an adjuvant. Various adjuvants used to increase theimmunological response include, but are not limited to, Freund's(complete and incomplete), mineral gels (e.g., aluminum hydroxide),surface active substances (e.g., lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, dinitrophenol, etc.), humanadjuvants such as Bacille Calmette-Guerin and Corynebacterium parvum, orsimilar immunostimulatory agents. If desired, the antibody moleculesdirected against HCELL can be isolated from the mammal (e.g., from theblood) and further purified by well known techniques, such as protein Achromatography to obtain the IgG fraction.

The term “monoclonal antibody” or “monoclonal antibody composition”, asused herein, refers to a population of antibody molecules that containonly one species of an antigen binding site capable of immunoreactingwith a particular epitope of HCELL. A monoclonal antibody compositionthus typically displays a single binding affinity for a particular HCELLglycoprotein with which it immunoreacts. For preparation of monoclonalantibodies directed towards a particular HCELL glycoprotein, orderivatives, fragments, analogs or homologs thereof, any technique thatprovides for the production of antibody molecules by continuous cellline culture may be utilized. Such techniques include, but are notlimited to, the hybridoma technique (see Kohler & Milstein, 1975 Nature256: 495-497); the trioma technique; the human B-cell hybridomatechnique (see Kozbor, et al., 1983 Immunol Today 4: 72) and the EBVhybridoma technique to produce human monoclonal antibodies (see Cole, etal., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss,Inc., pp. 77-96). Human monoclonal antibodies may be utilized in thepractice of the present invention and may be produced by using humanhybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA 80:2026-2030) or by transforming human B-cells with Epstein Barr Virus invitro (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCERTHERAPY, Alan R. Liss, Inc., pp. 77-96). Each of the above citations areincorporated herein by reference in their entirety.

According to the invention, techniques can be adapted for the productionof single-chain antibodies specific to a HCELL glycoprotein (see e.g.,U.S. Pat. No. 4,946,778). In addition, methodologies can be adapted forthe construction of F_(ab) expression libraries (see e.g., Huse, et al.,1989 Science 246: 1275-1281) to allow rapid and effective identificationof monoclonal F_(ab) fragments with the desired specificity for a HCELLglycoprotein or derivatives, fragments, analogs or homologs thereof.Non-human antibodies can be “humanized” by techniques well known in theart. See e.g., U.S. Pat. No. 5,225,539. Antibody fragments that containthe idiotypes to a HCELL glycoprotein may be produced by techniquesknown in the art including, but not limited to: (i) an F_((ab′)2)fragment produced by pepsin digestion of an antibody molecule; (ii) anF_(ab) fragment generated by reducing the disulfide bridges of anF_((ab′)2) fragment; (iii) an F_(ab) fragment generated by the treatmentof the antibody molecule with papain and a reducing agent and (iv) F_(v)fragments.

Additionally, recombinant anti-HCELL antibodies, such as chimeric andhumanized monoclonal antibodies, comprising both human and non-humanportions, which can be made using standard recombinant DNA techniques,are within the scope of the invention. Such chimeric and humanizedmonoclonal antibodies can be produced by recombinant DNA techniquesknown in the art, for example using methods described in InternationalApplication No. PCT/US86/02269; European Patent Application No. 184,187;European Patent Application No. 171,496; European Patent Application No.173,494; PCT International Publication No. WO 86/01533; U.S. Pat. No.4,816,567; U.S. Pat. No. 5,225,539; European Patent Application No.125,023; Better et al., (1988) Science 240:1041-1043; Liu et al. (1987)PNAS 84:3439-3443; Liu et al. (1987) J Immunol. 139:3521-3526; Sun etal. (1987) PNAS 84:214-218; Nishimura et al. (1987) Cancer Res47:999-1005; Wood et al. (1985) Nature 314:446-449; Shaw et al. (1988) JNatl Cancer Inst 80:1553-1559); Morrison (1985) Science 229:1202-1207;Oi et al. (1986) BioTechniques 4:214; Jones et al. (1986) Nature321:552-525; Verhoeyan et al. (1988) Science 239:1534; and Beidler etal. (1988) J Immunol 141:4053-4060. Each of the above citations areincorporated herein by reference in their entirety.

In one embodiment, methodologies for the screening of antibodies thatpossess the desired specificity include, but are not limited to,enzyme-linked immunosorbent assay (ELISA) and otherimmunologically-mediated techniques known within the art. In a specificembodiment, selection of antibodies that are specific to a particulardomain of a HCELL glycoprotein is facilitated by generation ofhybridomas that bind to the fragment of a HCELL glycoprotein possessingsuch a domain. Antibodies that are specific for a N-linked glycosylationsite, or derivatives, fragments, analogs or homologs thereof, are alsoprovided herein.

Anti-HCELL antibodies may be used in methods known within the artrelating to the localization and/or quantitation of a HCELL glycoprotein(e.g., for use in measuring levels of the HCELL glycoprotein withinappropriate physiological samples, for use in diagnostic methods, foruse in imaging the protein, and the like). In a given embodiment,antibodies for HCELL glycoproteins, or derivatives, fragments, analogsor homologs thereof, that contain the antibody derived binding domain,are utilized as pharmacologically-active compounds [hereinafter“Therapeutics”].

An anti-HCELL antibody (e.g., monoclonal antibody) can be used toisolate HCELL by standard techniques, such as affinity chromatography orimmunoprecipitation. An anti-HCELL antibody can facilitate thepurification of natural HCELL from cells and of recombinantly producedHCELL expressed in host cells. Moreover, an anti-HCELL antibody can beused to detect HCELL glycoprotein (e.g., in a cellular lysate or cellsupernatant) in order to evaluate the abundance and pattern ofexpression of the HCELL glycoprotein. Anti-HCELL antibodies can be useddiagnostically to monitor protein levels in tissue as part of a clinicaltesting procedure, e.g., to, for example, determine the efficacy of agiven treatment regimen. Alternatively, anti-HCELL antibodies are usedto treat or diagnosis leukemia. Detection can be facilitated by coupling(i.e., physically linking) the antibody to a detectable substance.Examples of detectable substances include various enzymes, prostheticgroups, fluorescent materials, luminescent materials, bioluminescentmaterials, and radioactive materials. Examples of suitable enzymesinclude horseradish peroxidase, alkaline phosphatase, β-galactosidase,or acetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin, and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ³⁵S or ³H.

HCELL Recombinant Expression Vectors and Host Cells

Another aspect of the invention pertains to vectors, preferablyexpression vectors, containing a nucleic acid encoding HCELLpolypeptides, fusion polypeptides, or derivatives, fragments, analogs orhomologs thereof. As used herein, the term “vector” refers to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments canbe ligated. Another type of vector is a viral vector, wherein additionalDNA segments can be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable ofdirecting the expression of genes to which they are operatively-linked.Such vectors are referred to herein as “expression vectors”. In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” can be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell, which means that the recombinant expression vectorsinclude one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, that is operatively-linked to thenucleic acid sequence to be expressed. Within a recombinant expressionvector, “operably-linked” is intended to mean that the nucleotidesequence of interest is linked to the regulatory sequence(s) in a mannerthat allows for expression of the nucleotide sequence (e.g., in an invitro transcription/translation system or in a host cell when the vectoris introduced into the host cell).

The term “regulatory sequence” is intended to includes promoters,enhancers and other expression control elements (e.g., polyadenylationsignals). Such regulatory sequences are described, for example, inGoeddel, Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990). Regulatory sequences include those thatdirect constitutive expression of a nucleotide sequence in many types ofhost cell and those that direct expression of the nucleotide sequenceonly in certain host cells (e.g., tissue-specific regulatory sequences).It will be appreciated by those skilled in the art that the design ofthe expression vector can depend on such factors as the choice of thehost cell to be transformed, the level of expression of protein desired,etc. The expression vectors of the invention can be introduced into hostcells to thereby produce proteins or peptides, including fusion proteinsor peptides, encoded by nucleic acids as described herein

The recombinant expression vectors of the invention can be designed forexpression of HCELL polypeptides or fusion polypeptides in prokaryoticor eukaryotic cells. For example, HCELL polypeptides or fusionpolypeptides can be expressed in bacterial cells such as Escherichiacoli, insect cells (using baculovirus expression vectors) yeast cells ormammalian cells. Suitable host cells are discussed further in Goeddel,Gene Expression Technology: Methods in Enzymology 185, Academic Press,San Diego, Calif. (1990). Alternatively, the recombinant expressionvector can be transcribed and translated in vitro, for example using T7promoter regulatory sequences and T7 polymerase.

Expression of proteins in prokaryotes is most often carried out inEscherichia coli with vectors containing constitutive or induciblepromoters directing the expression of either fusion or non-fusionproteins. Fusion vectors add a number of amino acids to a proteinencoded therein, usually to the amino terminus of the recombinantprotein. Such fusion vectors typically serve three purposes: (i) toincrease expression of recombinant protein; (ii) to increase thesolubility of the recombinant protein; and (iii) to aid in thepurification of the recombinant protein by acting as a ligand inaffinity purification. Often, in fusion expression vectors, aproteolytic cleavage site is introduced at the junction of the fusionmoiety and the recombinant protein to enable separation of therecombinant protein from the fusion moiety subsequent to purification ofthe fusion protein. Such enzymes, and their cognate recognitionsequences, include Factor Xa, thrombin and enterokinase. Typical fusionexpression vectors include pGEX (Pharmacia Biotech Inc; Smith andJohnson, 1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly,Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) that fuse glutathioneS-transferase (GST), maltose E binding protein, or protein A,respectively, to the target recombinant protein.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amrann et al., (1988) Gene 69:301-315) and pET 11d(Studier et al., Gene Expression Technology: Methods in Enzymology 185,Academic Press, San Diego, Calif. (1990) 60-89).

One strategy to maximize recombinant protein expression in E. coli is toexpress the protein in a host bacteria with an impaired capacity toproteolytically cleave the recombinant protein. See, e.g., Gottesman,Gene Expression Technology: Methods in Enzymology 185, Academic Press,San Diego, Calif. (1990) 119-128. Another strategy is to alter thenucleic acid sequence of the nucleic acid to be inserted into anexpression vector so that the individual codons for each amino acid arethose preferentially utilized in E. coli (see, e.g., Wada, et al., 1992.Nucl. Acids Res. 20: 2111-2118). Such alteration of nucleic acidsequences of the invention can be carried out by standard DNA synthesistechniques.

In another embodiment, the HCELL polypeptides or fusion polypeptidesexpression vector is a yeast expression vector. Examples of vectors forexpression in yeast Saccharomyces cerivisae include pYepSec1 (Baldari,et al., 1987. EMBO J. 6: 229-234), pMFa (Kurjan and Herskowitz, 1982.Cell 30: 933-943), pJRY88 (Schultz et al., 1987. Gene 54: 113-123),pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ (InVitrogenCorp, San Diego, Calif.).

Alternatively, HCELL polypeptides or fusion polypeptides can beexpressed in insect cells using baculovirus expression vectors.Baculovirus vectors available for expression of proteins in culturedinsect cells (e.g., SF9 cells) include the pAc series (Smith, et al.,1983. Mol. Cell. Biol. 3: 2156-2165) and the pVL series (Lucklow andSummers, 1989. Virology 170: 31-39).

In yet another embodiment, a nucleic acid of the invention is expressedin mammalian cells using a mammalian expression vector. Examples ofmammalian expression vectors include pCDM8 (Seed, 1987. Nature 329: 840)and pMT2PC (Kaufman, et al., 1987. EMBO J. 6: 187-195). When used inmammalian cells, the expression vector's control functions are oftenprovided by viral regulatory elements. For example, commonly usedpromoters are derived from polyoma, adenovirus 2, cytomegalovirus, andsimian virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells see, e.g., Chapters 16 and 17 ofSambrook, et al., Molecular Cloning: A Laboratory Manual. 2nd ed., ColdSpring Harbor Laboratory, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert, et al.,1987. Genes Dev. 1: 268-277), lymphoid-specific promoters (Calame andEaton, 1988. Adv. Immunol. 43: 235-275), in particular promoters of Tcell receptors (Winoto and Baltimore, 1989. EMBO J. 8: 729-733) andimmunoglobulins (Banerji, et al., 1983. Cell 33: 729-740; Queen andBaltimore, 1983. Cell 33: 741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci.USA 86: 5473-5477), pancreas-specific promoters (Edlund, et al., 1985.Science 230: 912-916), and mammary gland-specific promoters (e.g., milkwhey promoter; U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). Developmentally-regulated promoters are alsoencompassed, e.g., the murine hox promoters (Kessel and Gruss, 1990.Science 249: 374-379) and the α-fetoprotein promoter (Campes andTilghman, 1989. Genes Dev. 3: 537-546).

The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. That is, the DNA molecule isoperatively-linked to a regulatory sequence in a manner that allows forexpression (by transcription of the DNA molecule) of an RNA moleculethat is antisense to HCELL polypeptides or fusion polypeptides mRNA.Regulatory sequences operatively linked to a nucleic acid cloned in theantisense orientation can be chosen that direct the continuousexpression of the antisense RNA molecule in a variety of cell types, forinstance viral promoters and/or enhancers, or regulatory sequences canbe chosen that direct constitutive, tissue specific or cell typespecific expression of antisense RNA. The antisense expression vectorcan be in the form of a recombinant plasmid, phagemid or attenuatedvirus in which antisense nucleic acids are produced under the control ofa high efficiency regulatory region, the activity of which can bedetermined by the cell type into which the vector is introduced. For adiscussion of the regulation of gene expression using antisense genessee, e.g., Weintraub, et al., “Antisense RNA as a molecular tool forgenetic analysis,” Reviews-Trends in Genetics, Vol. 1(1) 1986.

Another aspect of the invention pertains to host cells into which arecombinant expression vector of the invention has been introduced. Theterms “host cell” and “recombinant host cell” are used interchangeablyherein. It is understood that such terms refer not only to theparticular subject cell but also to the progeny or potential progeny ofsuch a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

A host cell can be any prokaryotic or eukaryotic cell. For example,HCELL polypeptides or fusion polypeptides can be expressed in bacterialcells such as E. coli, insect cells, yeast or mammalian cells (such ashuman, Chinese hamster ovary cells (CHO) or COS cells). Other suitablehost cells are known to those skilled in the art.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid (e.g., DNA) into a host cell, including calcium phosphate orcalcium chloride co-precipitation, DEAE-dextran-mediated transfection,lipofection, or electroporation. Suitable methods for transforming ortransfecting host cells can be found in Sambrook, et al. (MolecularCloning: A Laboratory Manual. 2nd ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989),and other laboratory manuals.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest. Variousselectable markers include those that confer resistance to drugs, suchas G418, hygromycin and methotrexate. Nucleic acid encoding a selectablemarker can be introduced into a host cell on the same vector as thatencoding HCELL polypeptides or fusion polypeptides or can be introducedon a separate vector. Cells stably transfected with the introducednucleic acid can be identified by drug selection (e.g., cells that haveincorporated the selectable marker gene will survive, while the othercells die).

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, can be used to produce (i.e., express) HCELLpolypeptides or fusion polypeptides. Accordingly, the invention furtherprovides methods for producing HCELL polypeptides or fusion polypeptidesusing the host cells of the invention. In one embodiment, the methodcomprises culturing the host cell of invention (into which a recombinantexpression vector encoding HCELL polypeptides or fusion polypeptides hasbeen introduced) in a suitable medium such that HCELL polypeptides orfusion polypeptides is produced. In another embodiment, the methodfurther comprises isolating HCELL polypeptides or fusion polypeptidespolypeptide from the medium or the host cell.

Method of Identifying Stem Cells

The invention provides various methods of identifying and/or isolatingstem cells. A stem cell is a pluripotent cell of mesodermal, ectodermalor endodermal origin. Preferably, a stem cell is of mesodermal origin.More preferably, a stem cell is a hematopoiteic progenitor cell.

A stem cell is identified by contacting a test cell population with oneor more agents, e.g., a protein, polypeptide or small molecule, thatspecifically bind to a HCELL polypeptide. Preferably, an agent is anantibody or a fragment thereof. The antibody can be polyclonal ormonoclonal. For example, an agent is a HCELL antibody. Alternatively, anagent is an anti-CD44 antibody, or a HECA-452 antibody.

Specifically binding is meant that the interaction between cell and theagent is sufficent to form a complex. A cell/agent complex is detected.Presence of a complex indicates that the test cell is a stem cell. In analternative method, the stem cell is isolated from the test cellpopulation by removing the complex from the test cell population. Thecomplex can be separated from the test cell population by methods knownin the art, e.g. flow cytometry. Additionally, the stem cell can befurther isolated by separating the stem cell from the agent(s) bydisrupting the complex. The complex can be disrupted from example by ionchelation with dilute EDTA.

Alternately, a stem cell can be identified by providing a selectinpolypeptide, e.g. E-selectin or L-selectin, immobilized on a solid phasee.g., glass, plastic or membrane and contacting the solid phase with afluid sample containing a suspension of test cells. In some aspects thefluid sample is moving. By a moving fluid sample it is meant that thesample flows across the surface of the membrane in a unidirectionalmanner. Interactions between fluid sample in flow and immobilized ligandcan be examined under a wide range of defined flow conditions, rangingfrom static incubation through physiological levels of shear flow,static conditions and serial application of static and shear conditions,and into supraphysiologic shear levels. For example, shear flowconditions is a flow force greater than 0.6 dynes/cm². Alternatively,shear flow condition is a flow force at least 2.8 dynes/cm².Additionally, shear flow condition is a flow force of at least 9.0dynes/cm². In some aspects, the fluid move across the membrane such thatphysiological shear stress is achieved at the surface. The interactionbetween the solid phase and the cells is then determined. An interactionbetween the cells of the fluid sample and the solid phase indicates thatthe cell is a stem cell.

Also include in the invention is a method of isolating stem cells. Themethod includes providing a selectin polypeptide on a solid phase andcontacting the solid phase with a fluid sample containing a suspensionof cells. The cells that adhere to the solid phase are then recovered.Bound cells can be removed by any method known in the art (e.g., by ionchelation with dilute EDTA and/or application of high shear force).Bound cells recovered from the blot surface can thus be collected andanalyzed for phenotype or biological functions after elution. The ligandimmobilized on the matrix can be reused to compare interactions amongvarious cell groups or manipulated in situ to define characteristics ofthe cell population under investigation.

The interaction between the cells and the solid phase can be, e.g.,rolling, firm attachments or specific interaction. In some aspects, thespecific interaction is determined by the affinity coefficient. Forexample a specific interaction is an interaction that has a K_(d) is inthe range of 0.1 mM to 7 mM. Preferably, the K_(d) is greater than 1 mM.

A cell/agent interaction or alternately a cell/solid phase interactioncan be determined for example, by visual inspection under a microscope,colormetrically, fluorometrically, by flow cytometry or using aparrallel plate flow chamber assay. Alternatively, the interaction isanalyzed by labeling the cells, HCELL, polypeptide or the agent usingflorescent labels, biotin, enzymes such as alkaline phosphatase,horseradish peroxidase or beta-galactosidase, radioactive isotopes orother labels known in the art. The label can be added to the cells,HCELL polypeptide or the agent prior or subsequent to contacting thetest cell population with the agent. The membrane or solid phase canthen be subject to spectrophotometic or radiographic analysis toquantify the number interacting with the selectin polypetide of solidphase.

The invention also provide methods of treating cell disorders such ashematopoiteic disorders, cancer, or disorders amenable to treatment witha stem cell (i.e., stem cell therapy) such as myocardial infarction,Parkinson's disease, diabetes, congenital muscle dystrophies, stroke,genetic/congenital disorders (e.g., osteogenesis imperfecta) and liverdisorders in a mammal, e.g., human by administering the cells isolatedby the above described methods.

Methods of Increasing E-Selctin/L-Selectin Ligand Affinity

The invention provides a method of increasing the affinity of a cell forE-selectin and/or L-selectin, by providing a cell and contacting thecell with one or more agents that increases cell-surface expression oractivity a HCELL polypeptide on the cell.

The cell can be any cell capable of expressing HCELL polypeptide. Forexample the cell can be a stem cell (i.e., a pluripotent cell). A cellcan be of mesodermal, ectodermal or endodermal origin. Preferably, acell of mesodermal origin. More preferably a cell is a hematopoieticprogenitor cell. The cell population that is exposed to, i.e., contactedwith, the compound can be any number of cells, i.e., one or more cells,and can be provided in vitro, in vivo, or ex vivo.

Suitable agents can be, e.g., a polypeptide, a nucleic acid. For examplean agent can be a CD44, glycosyltransferase or glycosidase polypeptide,nucleic acid that encodes a CD44, glycosyltransferase or glycosidasepolypeptide or a nucleic acid that increases expression of a nucleicacid that encodes a include CD44, glycosyltransferase, or glycosidasepolypeptide and, and derivatives, fragments, analogs and homologsthereof. A nucleic acid that increase expression of a nucleic acid thatencodes a CD44, glycosyltransferase, or glycosidase polypeptideincludes, e.g., promoters, enhancers. The nucleic acid can be eitherendogenous or exogenous.

Suitable sources of nucleic acids encoding CD44 polypeptide include forexample the human CD44 nucleic acid (and the encoded protein sequences)available as GenBank Accession Nos. L05407 and CAA40133, respectively.Other sources include human CD44 nucleic acid and protein sequences areshown in GenBank Accession No. U35632 and P16079, respectively, and areincorporated herein by reference in their entirety. Suitable sources ofnucleic acids encoding glycosyltransferase polypeptide include forexample the human glycosyltransferase nucleic acid (and the encodedprotein sequences) available as GenBank Accession Nos. AJ276689 andCAB81779, respectively. Suitable sources of nucleic acids encodingglycosidase polypeptide include for example the human glycosidasenucleic acid (and the encoded protein sequences) available as GenBankAccession Nos. AJ278964 and CAC08178, respectively. The use of otherCD44, glycosyltransferase, or glycosidase polypeptides and nucleic acidsknow in the art are also within the scope of the invention.

The agent can be exposed to the cell either directly (i.e., the cell isdirectly exposed to the nucleic acid or nucleic acid-containing vector)or indirectly.

HCELL expression can be measured at the nucleic acid or protein level.Expression of the nucleic acids can be measured at the RNA level usingany method known in the art. For example, northern hybridizationanalysis using probes which specifically recognize one or more of thesesequences can be used to determine gene expression. Alternatively,expression can be measured using reverse-transcription-based PCR assays.Expression can be also measured at the protein level, i.e., by measuringthe levels of polypeptides encoded by the gene products. Such methodsare well known in the art and include, e.g., immunoassays based onantibodies to proteins encoded by the genes.

HCELL activity can be measured for example by L-selectin or E-selectinbinding activity

Methods of Increasing Engraftment Potential of a Cell

The invention provides methods of increasing the engraftment potentialof a cell.

By increasing engraftment potential is meant that the cell has a greatersurvival rate after transplantation as compared to an untreated cell.

A cell can of mesodermal, ectodermal or endoderamal origin. Preferably,the cell is a stem cell. More preferably the cell is of mesodermalorigin. For example, the cell is a hematopoietic progenitor cell.

Included in the invention is a method of increasing the engraftmentpotential of a cell by providing a cell and contacting said cell withone or more agents that increases cell-surface expression or activity ofa HCELL polypeptide on the cell. The invention further provides methodof increasing levels of engrafted stem cells in a subject, e.g., humanby administering to the subject an agent that increases cell-surface orexpression of the HCELL on one or more stem cells in the subject. Theagent can be administered in vivo, ex vivo or in vitro.

Also included in the invention is a method of increasing the engraftmentpotential of a cell population by providing a selectin polypeptide,e.g., E-selectin or L-selectin immobilized on a solid phase, phase e.g.,glass, plastic or membrane and contacting the solid phase with a fluidsample containing a suspension of test cells. In some aspects the fluidsample is moving. By a moving fluid sample it is meant that the sampleflows across the surface of the membrane in a unidirectional manner.Interactions between fluid sample in flow and immobilized ligand can beexamined under a wide range of defined flow conditions, ranging fromstatic incubation through physiological levels of shear flow, staticconditions and serial application of static and shear conditions, andinto supraphysiologic shear levels. For example, shear flow conditionsis a flow force greater than 0.6 dynes/cm². Alternately, shear flowcondition is a flow force at least 2.8 dynes/cm2. Additionally, shearflow condition is a flow force of at least 9.0 dynes/cm². In someaspects, the fluid move across the membrane such that physiologicalshear stress is achieved at the surface. The cells that adhere to thesolid phase are then recovered. Bound cells can be removed by any methodknown in the art (e.g., by ion chelation with dilute EDTA and/orapplication of high shear force). Bound cells recovered from the blotsurface can thus be collected and analyzed for phenotype or biologicalfunctions after elution. The ligand immobilized on the matrix can bereused to compare interactions among various cell groups or manipulatedin situ (as outlined below) to define characteristics of the cellpopulation under investigation. Further provided by the invention is amethod of increasing levels of engrafted stem cells in a subject, byadministering to subject a composition comprising the cells isolatedaccording to the above methods.

Suitable agents can be, e.g., a polypeptide, a nucleic acid. For examplean agent can be a CD44, glycosyltransferase or glycosidase polypeptide,nucleic acid that encodes a CD44, glycosyltransferase or glycosidasepolypeptide or a nucleic acid that increases expression of a nucleicacid that encodes a include CD44, glycosyltransferase, or glycosidasepolypeptide and, and derivatives, fragments, analogs and homologsthereof. A nucleic acid that increase expression of a nucleic acid thatencodes a CD44, glycosyltransferase, or glycosidase polypeptideincludes, e.g., promoters, enhancers. The nucleic acid can be eitherendogenous or as) exogenous.

Suitable sources of nucleic acids encoding CD44 polypeptide include forexample the human CD44 nucleic acid (and the encoded protein sequences)available as GenBank Accession Nos. LO5407 and CAA40133, respectively.Other sources include human CD44 nucleic acid and protein sequences areshown in GenBank Accession No. U35632 and P16079, respectively, and areincorporated herein by reference in their entirety. Suitable sources ofnucleic acids encoding glycosyltransferase polypeptide include forexample the human glycosyltransferase nucleic acid (and the encodedprotein sequences) available as GenBank Accession Nos. AJ276689 andCAB81779, respectively. Suitable sources of nucleic acids encodingglycosidase polypeptide include for example the human glycosidasenucleic acid (and the encoded protein sequences) available as GenBankAccession Nos. AJ278964 and CAC08178, respectively. The use of otherCD44, glycosyltransferase, or glycosidase polypeptides and nucleic acidsknow in the art are also within the scope of the invention.

The subject is preferably a mammal. The mammal can be, e.g., a human,non-human primate, mouse, rat, dog, cat, horse, or cow. Additionally,the subject suffers from or is at risk of developing a hematopoieticdisorder, (e.g, leukemia), cancer, or inflammatory disorders, includingchronic inflammatory disorders, e.g., (rheumatoid arthritis).

A mammal suffering from or at risk of developing a hematopoieticdisorder, (e.g, leukemia), cancer, inflammatory disorders, includinginflammatory chronic inflammatory disorders, (e.g., rheumatoidarthritis) can be identified by the detection of a known risk factor,e.g., gender, age, prior history of smoking, genetic or familialpredisposition, attributed to the particular disorder. Alternatively, amammal suffering from or at risk of developing a hematopoietic disorder,(e.g, leukemia), cancer, inflammatory disorders, including inflammatorychronic inflammatory disorders, (e.g., rheumatoid arthritis) can beidentified by methods known in the art to diagnosis a particulardisorder.

Methods of Treating Hematopoietic Disorders

The invention provides a method of treating hematopoietic disorders,(e.g., leukemia, aplastic anemia, non-Hodgkin's lymphoma, chronicmyeloid leukemia, multiple myeloma chronic lymphocytic leukemia, andvarious myelodysplastic syndromes), in a subject, by administering tothe subject an agent that decreases the cell-surface or expression ofthe HCELL polypeptide in the subject.

The invention further provides a method of treating hematopoieticdisorders in a subject by providing blood from the subject andcontacting the blood with one or more agents that specifically bind aHCELL polypeptide under conditions sufficient to form a complex betweenthe agent and a blood cell in the blood. Preferably the blood cell iscancerous. More preferably the blood cell is a leukemic blood cellComplex formation is detected and the complex is removed from the bloodthereby removing the cell. The blood is then reintroduced into thesubject.

Suitable agents include, a protein, polypeptide or small molecule, thatspecifically bind to a HCELL polypeptide. Preferably, an agent is anantibody or a fragment thereof. The antibody can be polyclonal ormonoclonal. For example, an agent is a HCELL antibody. Alternatively, anagent is an anti-CD44 antibody, or a HECA-452 antibody. Specificallybinding is meant that the interaction between cell and the agent issufficent to form a complex. The complex can be separated from the bloodby methods known in the art, e.g. flow cytometry.

Also included in the invention is a method of treating hematopoieticdisorders in a subject by providing blood from the subject and aselectin polypeptide, e.g., E-selectin or L-selectin immobilized on asolid phase e.g., glass, plastic or membrane and contacting the solidphase with a the blood. In some aspects the blood sample is moving. By amoving a blood sample it is meant that the sample flows across thesurface of the membrane in a unidirectional manner. Interactions betweenblood sample in flow and immobilized ligand can be examined under a widerange of defined flow conditions, ranging from static incubation throughphysiological levels of shear flow, static conditions and serialapplication of static and shear conditions, and into supraphysiologicshear levels. For example, shear flow conditions is a flow force greaterthan 0.6 dynes/cm². Alternatively, shear flow condition is a flow forceat least 2.8 dynes/cm2. Additionally, shear flow condition is a flowforce of at least 9.0 dynes/cm². In some aspects, the blood moves acrossthe membrane such that physiological shear stress is achieved at thesurface. The blood is then re-introduced into the subject.

Blood removal and re-infusion into a subject is accomplished byplasmapheretic techniques known in the art.

The invention further provides methods of treating hematopoieticdisorders in a subject by administering to the subject an agent thatspecifically binds to a HCELL glycoprotein. The agent can be for examplea polypeptide or small molecule. Preferably, the agent is a HCELLantibody. Specifically binding is meant that the interaction betweenHCELL glycoprotein and the agent is sufficent to form a complex. Uponcomplex formation, the agent may activate complement or mediate cellulartoxicity, (e.g., antibody dependent cellular cytotoxicity (ADCC)) orother direct immunologic effects.

Alternatively, a hematopoietic disorder can be treated by administeringto the subject an agent that includes a first and second domain. Thefirst domain includes a compound that pecifically binds to a HCELLglycoprotein. Preferably the first domain is a HCELL antibody orfragement thereof. The second domain includes a toxin linked by acovalent bond, e.g. peptide bond, to the first domain. A toxin includesany compound capable of destroying or selectively killing a cell inwhich it comes in contact. By “selectively killing” means killing thosecells to which the first domain binds. Examples of toxins include,Diphtheria toxin (DT) Pseudomonas exotoxin (PE), ricin A (RTA), gelonin,pokeweed antiviral protein, and dodecandron.

The first and second domains can occur in any order, and the agent caninclude one or more of each domain.

The subject is preferably a mammal. The mammal can be, e.g., a human,non-human primate, mouse, rat, dog, cat, horse, or cow.

Methods of Diagnosing or Determining the Susceptibility to a HematologicDisorder

The invention provides a method of diagnosing or determining thesusceptibility to a hematologic disorder in a subject by contacting asubject derived cell population with one or more agents thatspecifically bind a HCELL glycoprotein. Specifically binding is meantthat the interaction between cell and the agent is sufficent to form acomplex. A cell/agent complex is detected. Presence of a complexindicates the presence of or the susceptibility to a hematalogicdisorder in the subject

Hematologic disorders that can be detected by this method include forexample, anemia, neutropenia, thrombicytosis, myeloproliferativedisorders or hematologic neoplasms.

The subject is preferably a mammal. The mammal can be, e.g., a human,non-human primate, mouse, rat, dog, cat, horse, or cow.

Methods of Determining the Prognosis or Efficacy of Treatment of aHematologoc Disorder

The invention provides a method of determining the prognosis or efficacyof treatment of hematologic disorder in a subject by contacting asubject derived cell population with one or more agents thatspecifically bind a HCELL glycoprotein. Specifically binding is meantthat the interaction between cell and the agent is sufficent to form acomplex. A cell/agent complex is detected. Absence of a complexindicates favorable prognosis or efficacious treatment of thehematologic disorder in the subject. Presence of a complex indicates anunfavorable prognosis or non-efficacious treatment of the hematologicdisorder in the subject.

By “efficacious” is meant that the treatment leads to a decrease in thehematologic disorder in the subject. When treatment is appliedprophylactically, “efficacious” means that the treatment retards orprevents a hematologic disorder.

Hematologic disorders that can be detected by this method include forexample, example, anemia, neutropenia, thrombicytosis,myeloproliferative disorders or hematologic neoplasms.

The subject is preferably a mammal. The mammal can be, e.g., a human,non-human primate, mouse, rat, dog, cat, horse, or cow.

Method of Treating Inflammatory Disorders

The invention provides a method of treating inflammatory disorders in asubject, by administering to the subject an a HCELL glycoprotein orfragment thereof.

Inflammatory disorders include, for example, rheumatoid arthritis (RA),inflammatory bowel disease (IBD), and asthma.

Pharmaceutical Compositions Including HCELL Polypeptides FusionPolypeptides or Nucleic Acids Encoding Same

The HCELL polypeptides, or nucleic acid molecules encoding these fusionproteins and cells isolated according the methods of the invention,(also referred to herein as “Therapeutics” or “active compounds”) andderivatives, fragments, analogs and homologs thereof, can beincorporated into pharmaceutical compositions suitable foradministration. Such compositions typically comprise the nucleic acidmolecule, protein, cell or antibody and a pharmaceutically acceptablecarrier. As used herein, “pharmaceutically acceptable carrier” isintended to include any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like, compatible with pharmaceutical administration.Suitable carriers are described in the most recent edition ofRemington's Pharmaceutical Sciences, a standard reference text in thefield, which is incorporated herein by reference. Preferred examples ofsuch carriers or diluents include, but are not limited to, water,saline, finger's solutions, dextrose solution, and 5% human serumalbumin. Liposomes and non-aqueous vehicles such as fixed oils may alsobe used. The use of such media and agents for pharmaceutically activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active compound, use thereof inthe compositions is contemplated. Supplementary active compounds canalso be incorporated into the compositions.

The active agents disclosed herein can also be formulated as liposomes.Liposomes are prepared by methods known in the art, such as described inEpstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang etal., Proc. Natl. Acad. Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos.4,485,045 and 4,544,545. Liposomes with enhanced circulation time aredisclosed in U.S. Pat. No. 5,013,556.

Particularly useful liposomes can be generated by the reverse-phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol, and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid(EDTA); buffers such as acetates, citrates or phosphates, and agents forthe adjustment of tonicity such as sodium chloride or dextrose. The pHcan be adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation are vacuum dryingand freeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

In some embodiments, oral or parenteral compositions are formulated indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the subject to be treated; each unitcontaining a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on the uniquecharacteristics of the active compound and the particular therapeuticeffect to be achieved, and the limitations inherent in the art ofcompounding such an active compound for the treatment of individuals.

The nucleic acid molecules of the invention can be inserted into vectorsand used as gene therapy vectors. Gene therapy vectors can be deliveredto a subject by, for example, intravenous injection, localadministration (see, e.g., U.S. Pat. No. 5,328,470) or by stereotacticinjection (see, e.g., Chen, et al., 1994. Proc. Natl. Acad. Sci. USA 91:3054-3057). The pharmaceutical preparation of the gene therapy vectorcan include the gene therapy vector in an acceptable diluent, or cancomprise a slow release matrix in which the gene delivery vehicle isimbedded. Alternatively, where the complete gene delivery vector can beproduced intact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells that producethe gene delivery system.

Sustained-release preparations can be prepared, if desired. Suitableexamples of sustained-release preparations include semipermeablematrices of solid hydrophobic polymers containing the antibody, whichmatrices are in the form of shaped articles, e.g., films, ormicrocapsules. Examples of sustained-release matrices includepolyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate),or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919),copolymers of L-glutamic acid and y ethyl-L-glutamate, non-degradableethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymerssuch as the LUPRON DEPOT™ (injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate), andpoly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinylacetate and lactic acid-glycolic acid enable release of molecules forover 100 days, certain hydrogels release proteins for shorter timeperiods.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

Example 1 Identification of HECA-452-Reactive Membrane Glycoproteins onHuman Hematopoietic Cells

SDS-PAGE and Western blot analysis of HECA-452-reactive epitope(s) wasperformed on membrane proteins from various human hematopoietic celllines.

Cell Preparatation

Human hematopoietic cell lines (KG1a, HL6O, RPMI-8402 and K562) and theBM endothelial cell line BMEC-1 (Candal et al, 1996) were propagated inRPM1164O/10% FBS/1% penicillin-streptomycin (Life Technologies, Inc.,Grand Island, N.Y.). Fresh circulating leukemia blasts were isolated byFicoll-Hypaque (1.077-1.0800 g/ml) (ICN Biomedicals, Inc;, Aurora, Ohio)density gradient centrifugation from the peripheral blood of patientswhere they represented >80% of all circulating leukocytes. Normal humanBM cells were extracted from vertebral bodies of cadaveric organ donorsobtained with consent of donor families and through the cooperation ofthe New England Organ Bank (Newton, Mass.). BM mononuclear cells wereisolated by Ficoll-Hypaque (1.077-1.0800 g/ml) density gradientcentrifugation. BM cells were separated into CD34+ and lineage+/CD34−subpopulations using a negative cell selection StemSep™ human progenitorenrichment column (StemCell Technologies, Inc., Vancouver, BC Canada)or, alternatively, using positive selection for CD34+ cells or for othersubpopulations of bone marrow cells (monocytes (CD 14+), granulocytes(CD 15+), B cells (CD1 9+) or T cells (CD3+)) by immunomagnetic beadingseparation (Miltenyi Biotec, Auburn, Calif.). CD34+/CD44+, CD34+/CD44−and CD34−/CD44+ cell populations were isolated by cell sorting on aMoFlo apparatus (Cytomation) using fluorochrome-conjugated anti-CD34moAb (HPCA-2) (Becton-Dickinson) and anti-CD44 moAb (Hermes-1) (a giftfrom Dr. Brenda Sandmaier, Fred Hutchinson Cancer Research Center).

SDS-PAGE and Western Blots

Membrane preparations of HPCs were isolated as previously described(Sackstein and Dimitroff, 2000). For SDS-PAGE and Western blotting,membrane preparations were diluted in reducing sample buffer andseparated on 6-9% SDS-PAGE gels. Where indicated, membrane proteins werealso treated with N-glycosidase F (Roche Molecular Biomedicals) (8 U/mlfor 24 hr) or Vibrio cholerae neuraminidase (Roche MolecularBiochemicals) (0.1 U/ml H/H/Ca++ for 1 hr at 37° C.) as previouslydescribed (Sackstein and Dimitroff, 2000). Resolved membrane proteinswere transferred to Sequi-Blot™ PVDF membrane (Bio-Rad, Inc., Hercules,Calif.) and blocked with PBS/Tween-20/20% FBS for 1 hr at 4° C. Blotswere incubated with rat moAb HECA-452 (Pharmingen, San Diego, Calif.)(1.2 ug/ml PBS) or anti-PSGL-1 moAb 4H10 (Genetics Institute) for 1 hrat RT. Isotype control immunoblots using either rat 1 gM or mouse IgGwere performed in parallel to evaluate non-specific reactive proteins.After 3 washes with PBS/0.1% Tween-20, blots were incubated with eitherAP-conjugated rabbit anti-rat 1 gM Abs (1:400) or AP-conjugated goatanti-mouse IgG (1:8000) depending on the primary Ab. AP substrate,Western Blue® (Promega, Madison, Wis.), was then added to develop theblots.

As illustrated in FIG. 1A, numerous and distinct HECA-452-reactive bandswere detected on SDS-PAGE of membrane protein isolated from KG1a cells.Despite 10-fold less KG1a membrane protein loaded for analysis in theseblots compared with that of HL6O, RPMI-8402 or K562 cellular membraneprotein, KG1a cells contained markedly more HECA-452 staining displayedon several component protein bands. Only one broad band of approximately140 kDa was detected on HL60 cells, which corresponded to the monomerspecies of PSGL-1 by immunoblot (FIG. 1A). There were noHECA-452-reactive membrane proteins from RPMI-8402 or K562 cells eventhough PSGL-1 was detected on Western blots of these cells by usinganti-PSGL-1 antibody, 4H10 (Dimitroff et al., 2000). This findingsuggested that these cells lacked the appropriate HECA-452 bindingepitope and, at minimum, the E-selectin binding species of PSGL-1.

Example 2 Assessment of E-Selectin Binding of Human Hematopoietic CellsUnder Defined Shear Flow Conditions

To examine whether HECA-452 expression by human hematopoietic cell linescorrelated with E-selectin ligand activity, parallel-plate flow chamberstudies were performed to assess E-selectin binding under defined shearflow conditions.

Utilizing Cell Monolayers

E-selectin-mediated adhesive interactions, under defined shear stress,were examined between hematopoietic cell monolayers and suspensions ofCHO-E (Chinese hamster ovary cells stably transfected with full-lengthcDNA encoding human E-selectin) in flow. CHO-E and empty vectorconstructs (CHO-Mock) were maintained in MEM 10% FBS/1%Penicillin/Streptomycin (Life Technologies, Inc.) and HAM's F-12(Cellgro, Inc.)/5% FCS/1% Penicillin/Streptomycin, respectively. CHO-Ecell tethering and rolling on hematopoietic cell monolayers wasvisualized by video microscopy in real time using the parallel-plateflow chamber. Prior to experimentation, CHO-E cells were harvested with5 mM EDTA, washed twice in HBSS and suspended at lxiO7/ml in HBSS/10 mMHEPES/2 mMCaCl₂ (H/H/CC). Negative control groups were prepared byeither adding 5 mM EDTA to the H/H assay buffer (to chelate Ca++required for binding), treating CHO-E cells with anti-E-selectin Abs(clone 68-5H1 1; Pharmingen) (10 ug/ml), or using CHO-empty vectortransfectants (CHO-Mock cells). To prepare hematopoietic cellmonolayers >90% confluent, suspensions of cells (KG1a, HL60, K562, orRPMI 8402) at 2×106/ml RPMI164O without NaBicarbonate/2% FBS werecytocentrifuged in 6-well plates at 5×106/well and then fixed in 3%glutaraldehyde. Reactive aldehyde groups were blocked in 0.2M lysine,and plated cells were suspended H/H/Ca++. In parallel, cells were alsopretreated with either Vibrio cholerae neuraminidase (0.1μ/ml H/H/Ca++for 1 hr at 37° C.) or O-sialoglycoprotein endopeptidase (OSGE) (60μg/ml H/H/C++ for 1 hr at 37° C.; Accurate Chemicals, Westbury, N.Y.),respectively. Cell monolayers were placed in the parallel-plate flowchamber and CHO-E/Mock cells were perfused into the chamber. Afterallowing the CHO-E or CHO-Mock cells to come in contact with the cellmonolayers, the flow rate was adjusted to exert shear stress of 2.8dynes/cm². The number of CHO-E/Mock cell rolling on each monolayer wasmeasured in one frame of five independent fields under 10× magnificationfrom multiple experiments. A minimum of 3 experiments was performed andresults were expressed as the mean±standard deviation.

Alternatively, using freshly isolated human primary BMEC cultured aspreviously described (Rafii et al., 1994), BMEC from subcultures notolder than passage 5 were seeded at 105 cells/well in 6-well plates and,when 90-100% confluent, stimulated with IL-1α (40 U/ml) for 4 hr toupregulate the surface expression of E-selectin (expression of which wasmeasured by flow cytometric analysis). Live cultures were then placed inthe parallel-plate flow chamber and hematopoietic cells (107/ml inH/H/Ca++) were perfused into the chamber over the BMEC. Hematopoieticcell tethering and rolling was visualized at 2.8 dynes/cm2.Non-IL-1α-activated BMEC and IL-1a-activated BMEC treated with 10 μg/mlanti-E-selectin moAb (clone 68-5H11) served as controls for assessingspecificity of E-selectin-mediated adhesion. Cellular rolling wasquantified and expressed as described above.

Utilizing Immobilized Immunoprecipitates.

CD44 was immunoprecipitated from untreated cell lysates or from celllysates treated with N-glycosidase-F (0.8 U/ml) Vibrio choleraeneuraminidase (0.1 U/ml) or a-L-fucosidase (80 mU/ml), and PSGL-1 wasimmunoprecipitated from untreated cell lysates as previously described(Sackstein and Dimitroff, 2000; Dimitroff et al., 2000).

CD44 or PSGL-1 immunoprecipitates were spotted onto plastic petridishes, fixed in 3% glutaraldehyde and incubated in 0.2M lysine to blockunreactive aldehyde groups, and then non-specific binding was preventedby incubating in 100% FBS for 1 hr at RT. Fixed spots were also treatedwith Vibrio cholerae neuraminidase (0.1 U/ml assay medium), which wasoverlaid onto the spots and incubated at 37° C. for 1 hr. The proteindishes were placed in the parallel-plate flow chamber and CHO-E, CHO-P(CHO stably transfected with human cDNA encoding full lengthP-selectin), CHO-P cells treated with function blocking anti-P-selectinmoAb (clone AK-4; Pharmingen) (10 ug/ml), CHO-E cells treated withfunction blocking anti-E-selectin Abs (10 μg/ml) (clone 68-5H1 1) orMock transfectants were perfused into the chamber (2×10⁶/ml H/H/Ca++) ata flow rate of 0.2 ml/min until the cells were in contact with thesubstrate. The flow rate was then increased to achieve a shear stress of2.8 dynes/cm². The frequency of cells rolling per 100× magnificationfield was determined, and data were expressed as the mean±standarddeviation of 8 fields visualized from a minimum of 3 experiments.

Tethering and rolling of Chinese hamster ovary cells expressing highlevels of E-selectin (CHO-E) was observed on glutaraldehyde-fixedmonolayers of KG1a and HL60 cells at 2.8 dynes/cm² (FIG. 1B).Mock-transfected CHO cells (CHO-mock) displayed no rolling. CHO-E cellrolling was 2-fold higher on KG1a cells than on HL60 cells and wascompletely inhibited by adding 5 mM EDTA to the assay medium, bypreincubating CHO-E cells with anti-E-selectin Abs or by pretreatingKG1a or HL60 cells with Vibrio cholerae neuraminidase (which cleavesterminal sialic acids). There was no E-selectin ligand activity on celllines RPMI-8402 and K562, whose membrane proteins did not containHECA-452 epitopes. Surprisingly, the E-selectin ligand activity of KG1aand HL60 cells was not inhibited by incubating cells with HECA 452 at 50ug/ml and was not abrogated by pretreatment with OSGE (FIG. 1B), thoughOSGE bioactivity was confirmed by the disappearance of theOSGE-sensitive epitope, QBEND-10, on KG1a CD34 as measured by flowcytometry.

To analyze the interaction between human HPCs and naturally expressedE-selectin on cells of physiologic importance, freshly isolated human BMmicrovascular endothelial cells (BMEC) were utilized. Under physiologicshear flow conditions, KG1a and HL60 cell rolling on live monolayers ofIL-1a-activated BMECs were observed. Consistent with results fromexperiments using CHO-E cells to assess HPC E-selectin ligand activity,KG1a cells possessed a 4-fold greater capacity to roll on E-selectin onIL-1a-activated BMECs than HL60 cells, while RPMI-8402 and K562 cellspossessed minimal E-selectin ligand activity (FIG. 2). KG1a and HL60cellular E-selectin ligand activities were not observed onnon-IL-1α-activated BMEC or on IL-1a-activated BMEC treated with afunctional blocking anti-E-selectin moAb.

Example 3 Assessment of E-Selectin Glycoprotein Ligands from HumanHematopoetic Cells Using a Blot Rolling Assay

To examine the E-selectin ligand activity of all HECA-452-reactive KG1amembrane proteins, the adhesive interactions under shear flow betweenselectin-expressing whole cells and proteins immobilized on Westernblots was assessed (Dimitroff et al., 2000).

The blot rolling assay was performed as previously described (Dimitroffet al., 2000). Briefly, CHO-E, CHO-P or CHO-Mock transfectants wereisolated as described above, washed twice in HBSS and suspended at10⁷/ml in HBSS/10 mM HEPES/2 mMCaCl₂ (H/H/Ca++)/10% glycerol. Westernblots of HPC membrane preparations stained with HECA-452 were renderedtransparent by incubating them in H/H/Ca++/10% glycerol. The blots werethen placed in the parallel-plate flow chamber, and CHO transfectants(2×10⁶/ml) were perfused into the chamber. After allowing the cells tocome in contact with the blotting membrane, the flow rate was adjustedto exert a shear stress of 3.8 dynes/cm². The viscosity of 10% glyceroladhesion assay medium was considered in the calculation of shear stressvalues. The number of cells rolling on and between each immunostainedbanding region was quantified under 100× magnification within each fieldof view on the video monitor using molecular weight markers(Kaleidoscope Molecular Weight Markers, Bio-Rad Lab.; See Blue® fromNovex, Inc.) as guides to help align and visualize the apparentmolecular weights of the proteins of interest. A minimum of 3experiments was performed and results were expressed as the meanESD ofcell rolling/field at 100× magnification. Negative controls wereprepared by either adding 5 mM EDTA to the CHO-E H/H assay buffer tochelate Ca++ required for binding, pretreating CHO-E cells withanti-E-selectin Abs (clone 68-5H1 1; 10 μg/ml) or by assessing theability of CHO-Mock cells to interact with the immobilized proteins.

E-selectin ligand activity on HECA-452-stained bands at 100, 120, 140,190 and 220 kDa, but not at 74 kDa (FIG. 3A) was observed. CHO-mocktransfectants showed no interactions with any bands. Specificity forE-selectin was demonstrated by the abrogation of CHO-E cell rolling inthe presence of either 5 mM EDTA or anti-E-selectin functional blockingAbs. On HL60 cells, CHO-E cell rolling was observed only over the broad140 kDa HECA-452-immunostained band (i.e. PSGL-1/CLA). To determinewhether E-selectin binding determinants reside on N-glycans, KG1amembrane protein was treated with N-glycosidase-F. De-N-1glycosylatedproteins were resolved by SDS-PAGE and analysed for HECA-452 reactivityand E-selectin ligand activity. N-glycosidase-F treatment markedlydiminished HECA-452 staining (FIG. 3B) and also completely abolishedCHO-E cell rolling on all proteins on the blot, indicating that allglycoprotein E-selectin binding determinants on KG1a cells are displayedexclusively on N-glycans.

Example 4 Identification and Characterization of HCELL

The expression of HECA-452 epitopes on the 98 kDa KG1a membrane proteinafter sequential excisions from SDS-PAGE gels of varying acrylamidepercentage was followed. After each round of SDS-PAGE purification, the98 kDa protein maintained its capacity to support lymphocyte rolling inthe blot-based hydrodynamic flow assay. Following three rounds ofSDS-PAGE purification, a faint HECA-452 stained band was detected at˜190 kDa in addition to the 98 kDa band, suggesting that someaggregation of the 98 kDa protein may have occurred ciuripg theisolation procedure. The 98 kDa Coomassie-blue-stained gel fragment wasthen submitted for mass spectrometry analysis of trypsin-digestedpeptide fragments. The primary peptide map matched that of the standardform of CD44 previously shown to be expressed on KG1a cells. Usingmonoclonal Abs against CD44 (mouse IgG A3D8, or rat IgG Hermes-1) alongwith HECA-452, we immunoblotted the purified 98 kDa band following thethird gel isolation with either HECA-452, A3D8 or Hermes-1 (FIG. 8).Each antibody detected the 98 kDa species as well as the faint band at˜190 kDa, thought to represent aggregated protein. Correlation betweenthe HECA-452 and anti-CD44 monoclonal antibodies indicated that the KG1aglycoform of CD44 contains the HECA-452 carbohydrate determinant(s).

To distinguish KG1a cellular PSGL-1/CLA from the other HECA-452-reactivebands, blots of immunoaffinity-purified PSGL-1 and of total KG1amembrane protein were immunostained with either HECA-452 or anti-PSGL-1(FIG. 3C). KG1a cells express both the monomer and dimer isoforms ofPSGL-1, which represented the 140 and 220 kDa HECA-452-reactive proteins(FIG. 3C). Thus, the HECA-452 reactive bands at 100, 120 and 190 kDa,which support CHO-E cell rolling (FIG. 3A), corresponded to non-PSGL-1proteins.

To determine whether N-glycan-specific modifications of CD44 confer theE-selectin ligand activity, immunoaffinity-purified CD44 from KG1amembrane proteins were tested for its capacity to serve as an E-selectinligand in blot rolling assays. As shown in FIG. 3D, KG1a CD44 showedHECA-452 reactivity and possessed E-selectin ligand activity. Treatmentof CD44 with N-glycosidase-F markedly reduced HECA-452 reactivity andcompletely abrogated CHO-E cell rolling (FIG. 3D). Exhaustiveimmunoprecipitation of CD44 (3 rounds) resulted in the disappearance ofstainable CD44 molecule at 100 kDa (Hermes-1 immunoblot and HECA-452immunoblot) and of all E-selectin ligand activity at the 100 kDa and 190kDa bands (FIGS. 4A and 4B). Moreover, there was a 55% decrement inE-selectin ligand activity at the 120 kDa band after three rounds ofimmunoprecipitation (FIG. 4B). Notably, there was no difference inHECA-452 staining or in E-selectin ligand activity of the 140 kDamonomer species of PSGL-1 after removal of CD44.

To further explore the E-selectin ligand activity of CD44, we directlyimmunoprecipitated CD44 from human hematopoietic cell lines and analyzedCHO-E cell binding in the parallel-plate flow chamber. While CD44isolated from human hematopoietic cell lines HL60, K562 and RPMI 8402did not support any CHO-E cell rolling, KG1a CD44 exhibited E-selectinligand activity over a range of shear stress. Significantly greaterCHO-E cell rolling was observed on untreated KG1a CD44 than onN-glycosidase-F, on a-L-fucosidase-treated KG1a CD44, on Vibrio choleraeneuraminidase-treated KG1a membrane protein or on isotype Abimmunoprecipitates (control) (FIG. 5A). In addition, compared with amolecular equivalent amount of immunoprecipitated KG1a PSGL-1, CD44showed markedly greater E-selectin ligand activity at 2.8 dynes/cm²(p<O.OOO1) (FIG. 5A). However, at a lower shear stress of 0.6 dynes/cm²,CHO-E cell rolling on PSGL-1 was equivalent to that of CD44. These datasuggest that PSGL-1 and CD44 have overlapping contributions toE-selectin binding at low shear stress, but that CD44 engagement withE-selectin predominates at higher physiologic shear stress. Of note, CHOcells transfected with P-selectin rolled on KG1a PSGL-1, but not on KGIaCD44 (FIG. 5B), indicating that CD44 is not a P-selectin ligand (FIG.5B). In all experiments, negative control CHO cells (CHO-mocktransfectants and CHO-E or CHO-P cells treated with respective functionblocking anti-B- or P-selectin moAbs) did not tether and roll on anyproteins.

To determine whether CD44 naturally-expressed on normal human HPCsfunctions as an E-selectin ligand, we investigated the distribution ofHECA-452-reactivity and E-selectin ligand activity of CD44 expressed onearly CD34+ cells and more mature (CD34−/lineage+) human BM cells(including populations enriched for monocytes (CD14+), granulocytes (CD15+), and lymphocytes (B cells (CD19+) and T cells (CD3+)). Suprisingly,athough SDS-PAGE of Hermes-1 immunoprecipitates of KG1a cells revealsthree bands (100, 120 and 190 kDa), only a single 100 kDa CD44 wasimmunoprecipitated from both CD34+ and lineage+/CD34− cells, and onlyCD44 from CD34+ cells stained with HECA-452 and functioned as anE-selectin ligand (FIG. 6A). Similar results were obtained whether CD34+cells were enriched by negative selection or by positive selection. Evenwhen 10-fold excess lineage+ cell membrane protein was utilized for CD44immunoprecipitation, there was still neither HECA-452-staining of CD44nor E-selectin ligand activity of CD44 (FIG. 6A). Moreover, immobilizedon plastic; CD44 immunoprecipitated only from CD34+/CD44+ cellssupported CHO-E cell rolling whereas immunoprecipitated CD44 from CD34−cells did not possess any E-selectin ligand activity.

To further analyze the expression and structure of HECA-452-reactiveCD44 on human hematopoietic cells, expression of HCELL on nativeleukemic blasts was evaluated. Four major HECA-452 stained bands weredetected (74, 100, 140 and 190 kDa) from leukemic blasts of an acutemyelogenous leukemia (AML) (subtype M5) (FIG. 6B). HECA-452-staining wascompletely eliminated in the 100 kDa region following N-glycosidase-Ftreatment, while the 74 and 140 kDa bands had persistent staining andthe 100 kDa band stained at an apparently reduced molecular weight (FIG.6B). When immunoprecipitated CD44 from AML (M5) membrane protein wastreated with N-glycosidase-F, the HECA-452-reactivity as well as theE-selectin ligand activity was completely abolished (FIG. 6C). Similarto immunoprecipitated KG1a CD44, AML (M5) CD44 displayed a minor isoformat 120 kDa detected by HECA-452, but the major band and biologicallyactive protein was the 100 kDa CD44 isoform. CD44 was alsoimmunoprecipitated from blasts of an undifferentiated AML (MO), an AMLwithout maturation (Ml) and an atypical chronic myelogenous leukemia(CML) (bcr/abl−). Of note, expression of the HECA-452-reactive epitopeson immunoprecipitated CD44 directly correlated with the ability tosupport CHO-E cell rolling (FIG. 6D). The expression of CD44 (Hermes-1moAb) on these leukemias was equivalent (>90% positive cell staining byflow cytometry), further indicating that the ability to interact withE-selectin was dependent on the elaboration of HECA-452-reactiveglycosylations. Moreover, since CD44 is also expressed onnon-hematopoietic cells, we analyzed CD44 expressed on the human BMendothelial cell line BMEC-I. Though BMEC-1 expressed high levels ofCD44 (FIG. 6E), CD44 from these cells was not HECA-4S2-reactive and didnot possess any E-selectin ligand activity (FIG. 6D).

Example 5 Assessment of L-Selectin Glycoproetin Ligands from Human HeMatopoetic Cells Using a Blot Rolling Assay

To examine the L-selectin ligand activity of all HECA-452-reactive KG1amembrane glycoproteins, the adhesive interation under shear flow betweenselectin-expressing while cells and protein immobilized on Western Blotswas assessed.

L-selectin-expressing lymphocytes were isolated as previously described(Oxley, S. M. & Sackstein, R. (1994). Blood. 84:3299-3306; Sackstein,R., Fu, L. and Allen, K. L. (1997) Blood. 89:2773-2781), washed twice inHBSS and suspended at 2×10⁷/ml in HBSS/10 mM HEPES/2 mMCaCl₂(H/H/Ca)/10% glycerol. Cell lysate material is separated by SDS-PAGE andtransferred to PVDF under standard blotting conditions. Western blots ofKG1a membrane preparations stained with HECA-452, A3D8 or Hermes-1 wererendered transparent by incubating them in H/H/Ca⁺⁺/10% glycerol. Tostudy L-selectin-mediated adhesive interactions, the blots were placedin the parallel plate flow chamber and lymphocytes were perfused intothe chamber at a shear force of 2.3 dynes/cm² and cellular adhesiveinteractions are observed by video microscopy and analyzed in real time.

The number of lymphocytes rolling on and between each immunostainedbanding region was quantified from five independent fields under 200×magnification on the video monitor using molecular weight markers asguides to help align and visualize the proteins of interest. A minimumof 3 experiments were performed, and results were expressed as the meanof cell rolling/field. Negative controls were prepared by either adding5 mM EDTA to the lymphocyte H/H assay buffer to chelate Ca++ requiredfor binding, by using lymphocytes treated with PMA (50 ng/ml, whichinduces the cleavage of L-selectin (Oxley, S. M. & Sackstein, R. (1994).Blood. 84:3299-3306)), or by treating the lymphocytes with functionalblocking anti-L-selectin MoAbs (10 μg/ml) to verify the solecontribution of L-selectin.

Western blots of KG1a membrane proteins that were immunostained withHECA-452. (Oxley, S. M. & Sackstein, R. (1994). Blood. 84:3299-3306;Sackstein, R., Fu, L. and Allen, K. L. (1997). Blood. 89:2773-2781) wereperfused over the blots to assess for L-selectin ligand 9′ activity onresolved immunostained bands. Shear-dependent lymphocyte tethering androlling (at shear force of 2.3 dynes/cm²) on HECA-452-stained bands of98, 120 and 130 kDa that was L-selectin-dependent was observed. Toverify that the shear-dependent lymphocyte tethering and rolling wasL-selectin dependent, the assay was also performed in the presence of 5mM EDTA, and by pretreating the lymphocytes with either anti-L-selectinblocking monoclonal Abs or PMA (FIG. 7 D). There was no L-selectinligand activity displayed by any non-HECA-452-stained areas of the blot(FIG. 7 D). The HECA-452-stained 98 kDa band displayed the greatestL-selectin ligand activity (as much as 6-fold higher compared to otherbands), and this band is also the major N-glycan-bearing proteinexpressed on KG1a cells (FIG. 7C). Several of the HECA-452 reactive KG1abands did not possess L-selectin ligand activity suggesting that thestructural modification(s) associated with these HECA-452 reactiveproteins was not sufficient for L-selectin ligand activity. L-selectinligand activity was absent on Western blots of HL60, K562 and RPMI 8402membrane proteins, despite evidence of HECA-452-reactive bands.HECA-452-staining did not interfere with L-selectin-mediated lymphocyteadherence to the relevant immobilized KG1a proteins in hydrodynamic flowassays of Western blots.

Example 6 L-Selectin Ligand Activity of Immunoprecipitated KG1a CD44(HCELL)

Immunoblots of KG1a CD44 in total KG1a cell lysate using Hermes-1 moAbshowed a 98 kDa band, as well as 120 and 130 kDa bands which may reflectisoforms that were previously designated as CD44R2 and CD44R1,respectively (FIG. 9) (Dougherty, G. J., Lansdorp, P. M., Cooper, D. L.& Humphries, R. K. (1991). J. Exp. Med. 174:1-5). A faint signal at 190kDa that was detected by Hermes-1 and A3D8, which may reflect achondroitin sulfate-modified form of CD44 (FIG. 9) (Jalkanen, S. T.,Jalkanen, M., Bargatze, R., Tammi, M., & Butcher, E. C. (1988). J.Immunol. 141:1615-1623). To directly analyze whether this CD44 glycoformexhibited L-selectin ligand activity, KG1aCD44 from KG1a cells wasimmunoprecipitated with Hermes-1 moAb and then performed blot rollingassays on immunoblots of CD44 stained with either Hermes-1 or HECA-452.Surprisingly, Hermes-1-immunoprecipitated CD44 that was thenimmunoblotted with Hermes-1 displayed only the 98 kDa and 190 kDaspecies, but not the 120 and 130 kDa species. On the other hand,Hermes-1 immunoprecipitated CD44 that was immunoblotted with HECA-452illustrated not only the 98 kDa species, but also 120, 130 and 190 kDaspecies (FIG. 9). In all cases though, only the HECA-452-reactive,Hermes-1-reactive 98 kDa protein supported L-selectin ligandinteractions (FIG. 9).

Example 7 Dependence of N-Glycosylation for L-Selectin Ligand Activityand for Immunodetection by HECA-452

To examine the dependence of L-selectin ligand activity onN-glycosylation, immunoprecipitation of KG1a CD44 with Hermes-1 was alsoperformed on KG1a membrane proteins pretreated with N-glycosidase-F. Asdepicted in FIG. 10A, N-glycosidase-F treatment completely eliminatedHECA-452 staining of the 98 kDa species and abolished allL-selectin-mediated lymphocyte tethering and rolling on the blot. Ligandactivity over all molecular weight ranges in the N-glycosidase-F-treatedsample was assessed and some change in molecular weight withde-N-glycosylation of the glycoprotein was observed. Followingde-N-glycosylation, there were no bands demonstrating L-selectin ligandactivity.

To further analyze the L-selectin ligand activity of CD44, we performedStamper-Woodruff assays using immunoprecipitated CD44 or isotype control(rat IgG) immunoprecipitates of KG1a cells that was spotted onto glassslides as described (Sackstein, R. & Dimitroff, C. J. (2000). Blood. 96,2765-2774). Assays utilizing Vibrio cholerae neuraminidase-treated CD44,anti-L-selectin moAb (HRL-1 for rat lymphocytes; LAM1-3 for humanlymphocytes)-treated lymphocytes, or 5 mM EDTA co-incubation served asnegative controls. Immunoprecipitated, KG1a CD44 supportedL-selectin-mediated lymphocyte adherence (362±15 bound cells/field, 5fields counted, 3 slides per experiments, 2 experiments), whereas nobinding was observed with isotype control immunoprecipitate or withneuraminidase-treated immunoprecipitated KG1a CD44 orEDTA/anti-L-selectin Ab treatments (<10 cells bound cells/field).HECA-452 did not block lymphocyte adherence to isolated KG1a CD44,intact KG1a cells or to KG1a membrane proteins, despite inputconcentrations as high as 100 μg/ml. These results corroborated andconfirmed the data from the parallel-plate flow chamber studiesdescribed above.

Example 8 KG1a CD44 (HCELL) Functions as an L-Selectin Ligand in FreshlyIsolated Human Hematopoietic Cells

HCELL activity within normal marrow mononuclear cells was examined byStamper-Woodruff assays of sorted populations of CD34+/CD44+,CD34+/CD44−, CD34-CD44+ and CD34-CD44− cells. HCELL activity was absenton all CD44− subsets, but was present on >80% of CD34+/CD44+ cells andonly ˜1% of CD34-CD44+ cells. Because biochemical studies of CD44 onnormal human CD34+ bone marrow cells were limited by the difficulties inacquiring sufficient quantities of cells for such analysis, the HCELLactivity of CD44 isolated from blasts known to express HCELL activitywas examined (Sackstein, R. & Dimitroff, C. J. (2000). Blood. 96,2765-2774). Blasts from eleven leukemias: nine myelocytic (twoundifferentiated. (M0), two without maturation (M1), one with maturation(M2), two myelomonocytic (M4) and two monocytic (M5)), one acutelymphocytic (pre-B) and one biphenotypic were analyzed. With exceptionof one M0, all leukemic blasts displayed HCELL activity. The L-selectinligand activity of CD44 isolated from blasts of five of leukemiasdescribed above: one M0 shown to lack HCELL activity and the others withHCELL activity (the biphenotypic leukemia, the other MO, an M4, and anM5) was analazed. In blot rolling assays of total membrane protein,lymphocyte tethering and rolling was observed over a 98 kDa band in allleukemias expressing HCELL, but no rolling was observed on any membraneprotein from the M0 lacking HCLL activity. CD44 was immunoprecipitatedfrom each of these cells, and, similar to CD44 from KG1a cells, thepredominant isoform was a 98 kDa species. A representative example ofthese data, from an M4 leukemia, is shown in FIG. 10B. The requirementof N-glycosylated structures on CD44 for HECA-452 reactivity andL-selectin ligand activity was verified by pretreating the leukemiamembrane proteins with N-glycosidase-F (FIG. 10B). Conversely, thoughthe HCELL(−) M0 blasts possessed equivalent CD44 to that of the HCELL(+)leukemia specimens (as determined by flow cytometry and by Westernblotting using Hermes-1 Ab,), CD44 from these cells was notHECA-452-reactive and did not exhibit L-selectin ligand activity. Takentogether, these observations indicated that the CD44 glycoformexhibiting HCELL activity was not a unique feature of the KG1a cellline, but represented a physiologic modification of CD44 present inblasts of some subsets of human leukemias. These observations addedfurther evidence that component of N-glycans expressed on CD44.

Example 9 L-Selectin Ligand Activity of KG1a CD44 is Independent ofSulfation

To determine if CD44 on KG1a cells is sulfated, KG1a cell cultures weremetabolically labeled with [³⁵S]-SO₄. CD44 immunoprecipitated from thesecells was indeed sulfated (FIG. 11A). To test whether sulfation wascritical for L-selectin ligand activity of CD44, KG1a cells werepretreated with 0.1% bromelain, a protease that eliminates all KG1a HCLLactivity (Sackstein, R. & Dimitroff, C. J. (2000). Blood. 96, 2765-2774)and also removes CD44 from the cell surface (Hale, L. P. & Haynes, B. F.(1992). J. Immunol 149:3809-3816). Following bromelain digestion ofKG1a, re-expression of HCELL requires de novo protein synthesis, andprotein synthesized in the presence of chlorate (a metabolic inhibitorof both protein and carbohydrate sulfation) is non-sulfated (Sackstein,R., Fu, L. and Allen, K. L. (1997). Blood. 89:2773-2781). Therefore,KG1a was treated with bromelain and confirmed removal of CD44 by flowcytometry. The KG1a cells were cultured in the absence or presence of 10mM sodium chlorate for 24 hr and metabolically radiolabeled the cellsfor the last 8 hr of incubation with [³⁵S]-SO₄ in sulfate-deficientCRCM-30 medium. As illustrated in FIG. 11A, the incorporation of[³⁵S]-SO₄ into immunoprecipitable CD44 (Hermes-1) was completelyinhibited in chlorate-treated cells. This effect of chlorate wasspecific for sulfate incorporation and not a general inhibition of CD44protein synthesis, as [³⁵S]-methionine/cysteine metabolic radioloabelingof CD44 was identical in chlorate- and non-chlorate-treated cellpopulations.

Blot rolling assays were then performed on CD44 immunoprecipitated fromcontrol and chlorate-treated cells. The L-selectin ligand activity ofsulfated and non-sulfated CD44 (FIG. 11B). These experimental dataconfirmed the results of our previous studies that demonstrate thesulfation-independence of HCLL activity (Sackstein, R., Fu, L. andAllen, K. L. (1997). Blood. 89:2773-2781). Surprisingly, recognition ofsulfate-free CD44 with HECA-452 was not prevented (FIG. 11B). These datashow that sulfation was not a critical feature of the epitope on KG1aCD44 recognized by the HECA-452 monoclonal antibody.

Example 10 Differential L-Selectin Bonding Activities of HumanHematopoietic Cell L-Selectin Ligands, HCELL and PSGL-1

A. General Methods

Cell, Antibodies and Enzymes Human hematopoietic cell lines KG1a(myelocytic leukemic, HCELL+/PSGL-1+), HL60 (promyelocytic leukemia,HCELL−/PSGL-1+), RPMI 8402 (lymphocytic leukemia, HCELL−/PSGL-1+) andK562 (erythrocytic leukemia, HCELL−/PSGL-1−), and circulating blastsfrom a de novo acute myeloid leukemia (AML) without maturation (M1)(HCELL⁺/PSGL-1⁺) were maintained in RPMI-1640/10% FBS/1%Penicillin-Streptomycin (Life Technologies, Inc.). Chinese hamster ovary(CHO) cells transfected with full length cDNA encoding P-selectin(CHO-P; clone E4I) and CHO-empty vector (CHO-Mock) were obtained fromRobert C. Fuhlbrigge (Harvard Medical School), and maintained in MEM/10%FBS/1% Penicillin/Streptomycin (Life Technologies, Inc.) and HAM'sF-12/5 mM Glutamine/5% FCS/1% Penicillin/Streptomycin. Human lymphocytes(PBMC) were prepared from whole blood as previously described (Oxley, S.M. and Sackstein, R. (1994) Blood 84(10), 3299-3306). Rat thoracic ductlymphocytes (TDL) that express high levels of L-selectin, whichfunctions identically to human lymphocyte L-selectin, were obtained bycannulation of the rat thoracic duct as previously described (Oxley, S.M. and Sackstein, R. (1994) Blood 84(10), 3299-3306). Human neutrophilswere prepared from peripheral whole blood, collected in acid citratebuffer/0.6% dextran and red cells were allowed to separate under gravityfor 30 min. at RT. Leukocyte rich plasma was diluted 1:1 with PBSwithout Ca⁺⁺/Mg⁺⁺ and granulocytes were pelleted by Ficoll-Hypaque(1.077-1.0800 g/ml) density gradient centrifugation. To lyse residualred cells in cell pellets, cells were exposed to hypotonic solution for30 sec. and then neutralized with hypertonic NaCl. This procedureresulted in a >98% enrichment of granulocytes. Circulating leukemicblasts from a patient with an acute myeloid leukemia without maturation(M1) were isolated by Ficoll-Hypaque (1.077-1.0800 g/ml) densitygradient centrifugation from peripheral blood.

Anti-PSGL-1 monoclonal Ab PL-1 and PL-2, FITC-anti-CD45, and anti-CD34QBEND Abs were purchased from Coulter-Immunotech, Marseilles, France.Anti-PSGL-1 monoclonal Ab PSL-275 was a gift from Dr. Ray Camphausen(Genetics Institute, Cambridge, Mass.). Anti-sialyl Lewis X monoclonalAb (CSLEX-1), anti-CD44 (clone L178) and anti-CD43 antibodies werepurchased from Becton Dickinson, San Jose, Calif. Anti-CD44 moAbHermes-1 (rat IgG2a) was originally characterized by Jalkanen et al.(Jalkanen, S. T., Bargatze, R. F., Herron, L. R. and Butcher, E. C.(1986) Eur. J. Immunol. 16, 1195-1202). Rat monoclonal Ab anti-human CLA(HECA-452), anti-rat L-selectin (HRL-1; ligand blocking antibody),anti-human P-selectin (clone AK-4) and anti-human L-selectin (LAM1-3)were purchased from Pharmingen, San Diego, Calif. Allfluorochrome-conjugated secondary antibodies and isotype controls wereobtained from Zymed, San Francisco, Calif.

OSGE was purchased from Accurate Chemicals, Westbury, N.Y., and Vibriocholera neuraminidase and N-glycosidase-F was obtained from RocheMolecular Biochemicals, Indianapolis, Ind. Cobra venom metalloprotease,mocarhagin (Spertini, O., Cordey, A. S., Monai, N., Giuffre, L. andSchapira, M. (1996) J. Cell Biol. 135(2), 523-531; De Luca, M., Dunlop,L. C., Andrews, R. K., Flannery, J. V., Ettling, R., Cumming, D. A.,Veldman G. M. and Berndt, M. C. (1995) J. Biol. Chem. 270(45),26734-26737), was a gift from Dr. Ray Camphausen (Genetics Institute,Cambridge, Mass.). The metabolic inhibitor, tunicamycin, and all otherchemicals were purchased from Sigma, Inc. (St. Louis, Mo.).

Flow Cytometric Analysis. Flow cytometric analysis was performed onhuman HCs utilizing both direct and indirect immunofluorescence stainingapproaches. All cells utilized for these experiments were washed twicewith cold PBS/2% FBS, suspended at 10⁷/ml PBS/1% FBS. Primaryantibodies, anti-CLA, -CD15s, -CD34, -CD43, -CD44, -CD62L, -PSGL-1(PSL-275, and PL-1) along with the appropriate isotype-matched controlantibodies were incubated with the cells for 30 min. on ice. Followingtwo washes with PBS/2% FBS, cells were resuspended in PBS/1% FBS andincubated with respective fluorochrome-conjugated secondary antibodies(2 μl) for 30 min. on ice. Cells were washed twice with PBS/2% FBS,resuspended in PBS and flow cytometry was performed on a FACScanapparatus equipped with an argon laser tuned at 488 nm (BectonDickinson).Radiolabeling of Human HC Membrane Proteins, SDS-PAGE and WesternBlotting. Cell cultures (1-2×10⁶ cells/ml) were incubated with[³⁵S]EasyTag™-L-Methionine (150 μCi/ml) (NEN™, Boston, Mass.) inRPMI-1640 medium without methionine for 8 hr before membrane proteinpreparation. Membrane proteins were prepared as previously described.For SDS-PAGE and Western blotting, membrane preparations orimmunoprecipitates were diluted in reducing sample buffer and separatedon 7% SDS-PAGE gels. For autoradiography, gels resolving [³⁵S]-labeledimmunoprecipitates were dried and exposed to Kodak BioMax MR film(Fisher Scientific) for 3 hr. For Western blotting, resolved membraneproteins were transferred to Sequi-Blot™ PVDF membrane (Bio-Rad, Inc.,Hercules, Calif.) and blocked with PBS/0.1% Tween-20/60% FBS for 2 hr at4° C. Blots were incubated with rat IgM anti-human CLA HECA-452 (1.2μg/ml), rat IgG anti-human CD44 Hermes-1 (1 μg/ml) or anti-human PSGL-1Ab PL-2 (1 μg/ml) for 1 hr at RT. Isotype control immunoblots usingeither rat IgM, rat IgG or mouse IgG were performed in parallel toevaluate non-specific reactive proteins. After three washes withPBS/0.1% Tween-20, blots were incubated with the respective secondaryAb, AP-conjugated rabbit anti-rat IgM Abs (1:400), goat anti-rat IgG orgoat anti-mouse IgG (1:8000) (Zymed Labs. Inc., San Francisco, Calif.).AP substrate, Western Blue® (Promega, Madison, Wis.) was then added todevelop the blots.

Hydrodynamic Parallel-Plate Flow Chamber Analysis

L-Selectin-Mediated Adhesive Interactions

Using the parallel-plate flow chamber under defined shear stressconditions, L-selectin-mediated adhesive interactions between human HCsand L-selectin naturally expressed on leukocytes (awrence, M. B.,McIntire, L. V. and Eskin, S. G. (1987) Blood 70(5), 1284-1290) wasstudied. Leukocyte tethering and rolling on human HC monolayers wasvisualized by video microscopy in real time using the parallel-plateflow chamber prepared in the following manner. Prior to experimentation,leukocytes were washed twice in HBSS and then suspended at 10⁷/ml inHBSS/10 mM HEPES/2 mMCaCl₂ (H/H/Ca⁺⁺). Negative control groups wereprepared by treating cells with PMA (50 ng/mlH/H/Ca++ for 1 hr at 37°C.) to induce the cleavage of L-selectin from the cell surface, bypretreating with moAb HRL-1 (10 μg/ml) to block L-selectin binding, orby incubating with 5 mM EDTA to chelate Ca⁺⁺ required for L-selectinbinding. To prepare human HC monolayers (100% confluent), suspensions ofHCs (KG1a, HL60, RPMI 8402, K562) at 2×10⁶/ml RPMI-1640 without Na+Bicarbonate/2% FBS were seeded in 6-well plates at 5×10⁶/well,centrifuged to layer cells then fixed in 3% glutaraldehyde. Reactivealdehyde groups were blocked in 0.2M lysine, and plated cells weresuspended 1-1/H/Ca⁺⁺. To assess the dependence of binding by sialic acidresidues on L-selectin ligands, cells were pretreated with Vibriocholerae neuraminidase (0.1 U/ml H/H/Ca⁺⁺ for 1 hr at 37° C.). Toexamine the contribution of sialomucins (including PSGL-1) and PSGL-1alone, OSGE (60 μg/ml H/H/Ca⁺⁺ for 1 hr at 37° C.) or mocarhagin (10ug/ml for 20 min at 37° C.) treatments were performed, respectively.Furthermore, since HCELL is expressed on KG1a cells and sialylatedN-glycosylations on HCELL are critical for L-selectin ligand activity(Sackstein, R. and Dimitroff, C. J. (2000) Blood 96, 2765-2774) thecontribution of HCELL on KG1a cells was distinguished by first cleavingall of the sialic acid residues from the cell surface with Vibriocholerae neuraminidase (0.1 U/ml for 1 hr at 37° C.) and then incubatingthe cells with a metabolic FIO inhibitor of N-glycosylation, tunicamycin(15 μg/ml for 24 hr at 37° C., 5% CO₂), to prevent de novo synthesis ofN-glycans (i.e., HECA-452 epitopes on CD44/HCELL). Neuraminidasepretreatment removed all of the residual HCELL activity from the cellsurface, and therefore, this treatment approach allowed for theassessment of newly synthesized HCELL on the cell surface. HC cytospinpreparations were prepared in multi-well plates as described above. Theparallel-plate flow chamber was placed on top of the cell monolayers andleukocytes were perfused into the chamber. After allowing the leukocytesto contact the cell monolayers at a shear stress of 0.5 dynes/cm² (atwhich they do not engage in adhesion events), we adjusted the flow rateaccordingly to exert shear stress from 1 to >30 dynes/cm². The number ofleukocytes rolling in one frame of five independent fields under 200×magnification at shear stress of 0.2, 0.4, 0.8, 2.2, 4.4, 8.8, 17.6 and26.4 dynes/cm² were quantified. A minimum of 3 experiments was performedover the entire range of shear stress and results were expressed as themean±standard deviation.

P-selectin-Mediated Adhesive Interactions. In these experiments,glutaraldehyde-fixed HC monolayers were prepared in 6-well plates asdescribed above, and, where indicated, cells were pretreated withmocarhagin (10 μg/ml) for 30 min. and washed extensively with RPMI1640without Na+ Bicarbonate/2% FBS prior to fixation. To study P-selectinadhesive interactions, confluent CHO cells stably expressing full-lengthP-selectin (CHO-P) or empty vector (CHO-Mock) were released from flaskswith 5 mM EDTA, washed extensively in H/H/Ca⁺⁺ and resuspended at2×10⁶/ml for utilization in the parallel-plate flow chamber. P-selectinexpression on CHO-P cells was confirmed by flow cytometric analysis.Cell suspensions containing 5 mM EDTA or anti-P-selectin moAbs (10 μg/mlfor 30 min. on ice) were utilized as negative controls to confirmcalcium-dependent binding. Cells were perfused into the chamber andallowed to fall onto cell monolayers before commencing the assessment ofP-selectin adhesion at 0.2, 0.4, 0.8, and 2.2 dynes/cm². Cellulartethering and rolling was visualized at 100× magnification andquantified and analyzed as described above.

Stamper-Woodruff Assay.

L-Selectin-Mediated Lymphocyte Adherence to HCELL and to PSGL-1. Molarequivalents of immunoaffinity purified HCELL or PSGL-1 (0.75 μg ofreduced HCELL (100 kDa) and 1 μg of fully reduced PSGL-1 (140 kDa)) werespotted onto glass slides and allowed to dry. These protein spots werethen fixed in 3% glutaraldehyde, unreactive aldehyde groups were blockedin 0.2M lysine and slides were kept in RPMI-1640 withoutNaBicarbonate/2% FBS until ready for testing. Lymphocytes (10⁷/mlRPMI-1640 without NaBicarbonate/5% FBS) were overlayed onto these fixedimmunoprecipitates and incubated on an orbital shaker at 80 rpm for 30min. at 4° C. The number of adherent lymphocytes was quantified by lightmicroscopy using an ocular grid under 100× magnification (a minimum of 5fields/slide, 2 slides/experiment, and 3 separate experiments). Datawere presented as the mean number of bound lymphocytes±standarddeviation. KG1a CD34 and L-selectin control immunoprecipitates were alsotested in this manner. To verify the dependence for sialic acid,glutaraldehyde-fixed spots were treated with Vibrio choleraeneuraminidase (0.1 U/ml RPMI-1640 without NaBicarbonate/2% FBS for 1 hrat 37° C.). In addition, to verify that cellular adhesion was dependenton L-selectin, all assays included negative controls, in whichlymphocytes were treated with PMA (50 ng/ml for 30 min at 37° C.) orfunctional blocking antibodies (anti-rat-L-selectin moAb HRL-1 oranti-human L-selectin moAb LAM1-3; 10 μg/ml), or lymphocyte suspensionscontained 5 mM EDTA.

L-Selectin-Mediated Lymphocyte Adherence to Human HCs. For analysis ofcellular L-selectin ligand activity of human HCs, cytospin preparationsof KG1a, HL60, RPMI-8402 and K562 cells, and of de novo leukemia blastswere fixed in 3% glutaraldehyde, blocked in 0.2M lysine and overlayedwith lymphocytes (10⁷ cells/ml RPMI-1640 without Na+ Bicarbonate/5% FBS)on an orbital shaker at 80 rpm for 30 min. at 4° C. Slides were thencarefully washed with PBS, and bound lymphocytes were fixed in 3%glutaraldehyde. All assays included negative controls as describedabove. Data were presented as the mean (±S.D.) number of boundlymphocytes at 100× magnification from a minimum of 5 fields/slide induplicate slides from 3 separate experiments.

B. HCELL is Capable of Engaging with L-Selectin Over a Wide Range ofShear Stress.

The goal of this study was to assess the capability of HCELL and PSGL-1on human HCs to support shear-dependent L-selectin binding activity overa range of shear stress. The L-selectin binding characteristics of eachof these molecules were analyzed by performing shear-based adherenceassay systems using human HCs that expressed HCELL and/or PSGL-1: KG1a(HCELL+/PSGL-1+), HL60 (HCELL−/PSGL-1+), RPMI 8402 (HCELL−/PSGL-1+),K562 (HCELL−/PSGL-1−) and a de novo acute myeloid leukemia (AML) withoutmaturation (M1) (HCELL⁺/PSGL-1⁺) (Table 2) (Oxley, S. M. and Sackstein,R. (1994) Blood 84(10), 3299-330: Sackstein, R., Fu, L. and Allen, K. L.(1997) Blood 89(8), 2773-2781; Sackstein, R. and Dimitroff, C. J. (2000)Blood 96, 2765-2774.)

TABLE 1 Flow Cytomatric Analysis of Sialoglycoconjugates onHematopoietic Cell Lines Hematopoietic % Positive Cell Staining* CellLines¹ CD15s CD34 CD43 CD44 CD45 PSGL-1 CLA CD62L KG1a (Myeloid) ++++++++ ++++ ++++ ++++ ++++ ++++ +++ K562 (Erythroid) − − ++++ + − − − −RPM 8402 (Lymphoid) − ++++ ++++ ++++ ++++ +++ − ++++ HL-60 (Promeyloid)+++ − ++++ ++++ ++++ ++++ ++++ − ¹All cell lines were maintained inRPM1640/10% FBS/1% penicillin-streptomycin and grown to confluency (1-2× 10⁶/ml). Cells were then isolated, washed in PBS/2% FBS, suspended at10⁷/ml PBS/1% FBS and stained/analyzed as described in the Materials andMethods. *Percent positive cell staining indicates the number of cellsthat stain greater than negative control cell staining (autofluorescenceor FITC-conjugated secondary Ab alone groups). The percentages arerepresented as follows − = 0-19%, + = 20-39, ++ = 40-59%, +++ = 60-79%and ++++ = 80-100%

Using the parallel-plate flow chamber under defined hydrodynamic shearstress, L-selectin-mediated tethering and rolling of leukocytes overglutaraldehyde-fixed human HC monolayers was observed. All experimentsincluded negative controls to verify the sole contribution of L-selectinin mediating cell-cell adherence. A shear stress threshold of ˜0.5dynes/cm² was required for L-selectin-mediated adhesive interactions inthis system as previously demonstrated (Lawrence, M. B., Kansas, G. S.,Kunkel, E. J. and Ley, K. (1997) J. Cell Biol. 136(3), 717-727; Finger,E. B., Puri, K. D., Alon, R., Lawrence, M. B., von Andrian, U. H. andSpringer, T. A. (1996) Nature 379(6562):266-269.) (FIG. 12A). Afterreaching this level of shear stress, leukocyte tethering and rolling wasenumerated over a broad shear stress range. L-selectin-dependent humanlymphocyte and rat TDL rolling on HL60 cells at a shear stress range of0.4 dynes/cm² to a maximum of 10 dynes/cm² was observed (FIG. 12A).However, there was no evidence of lymphocyte rolling on K562 cells, and,despite high PSGL-1 expression, RPMI 8402 cells also did not displayL-selectin ligand activity (FIG. 12A and Table 2). In contrast,L-selectin-mediated human lymphocyte and rat TDL rolling on KG1a cellmonolayers was observed at shear stress levels in excess of 26dynes/cm², whereas, on HL60 cells, human lymphocyte/TDL rolling wasabsent past 17 dynes/cm² (FIG. 12A). In addition, the frequency ofrolling lymphocytes on KG1a cells was up to a 5-fold greater over theentire range of shear stress that supported L-selectin-mediated rollingon HL60 cells (FIG. 12A). The disparity between the high L-selectinligand activity on KG1a cells and low activity on HL60 cells was alsoobserved by using human neutrophils, which expressed equivalent levelsof L-selectin by flow cytometric analysis: KG1a cells supported 4-foldgreater L-selectin-mediated neutrophil rolling than that on HL60 cells(FIG. 12B). These data show that KG1a cells possess greater capacity tosupport L-selectin mediated leukocyte adherence over a broader range ofshear stress and that L-selectin natively expressed on lymphocytes or onneutrophils exhibits comparable binding activity to HCELL or to PSGL-1expressed on human HCs.

To distinguish the contribution of PSGL-1 activity from HCELL activityon KG1a cells, enzymatic digestion of cells with OSGE or mocarhagin, orincubated cells with a functional blocking Ab PL-1 that both renderPSGL-1 incapable of binding to L-selectin was performed (Spertini, O.,Cordey, A. S., Monai, N., Giuffre, L. and Schapira, M. (1996) J. CellBiol. 135(2), 523-531; Guyer, D. A., Moore, K. L., Lyman, E. B.,Schammel, C. M. G., Rogelj, S., McEver, R. P. and Sklar, L. A. (1996)Blood 88, 2415-2421; Tu. L., Chen, A., Delahunty, M. D., Moore, K. L.,Watson, S. R., McEver, R. P. and Tedder, T. F. (1996) J. Immunol. 157,3995-4004; De Luca, M., Dunlop, L. C., Andrews, R. K., Flannery, J. V.,Ettling, R., Cumming, D. A., Veldman G. M. and Berndt, M. C. (1995) J.Biol. Chem. 270(45), 26734-26737 14-16). Alternatively, to distinguishthe contribution of HCELL activity on KG1a cells (which is expressedexclusively on sialylated N-glycans), KG1a cells and blasts from the denovo leukemia were pretreated with neuraminidase then incubated intunicamycin. Accordingly, L-selectin ligand activity of KG1a cells wasresistant to enzymatic digestion with OSGE or mocarhagin, and PL-1antibody treatments (Table 3). However, KG1a L-selectin ligand activitywas eliminated following neuraminidase digestion, re-expression ofligand activity was markedly reduced following tunicamycin treatment,while ligand activity of cells treated with DMSO alone (control)returned to baseline levels (p<0.001) (Table 3). These data show thatN-glycan-dependent HCELL is the primary mediator of L-selectin bindingon KG1a cells. In contrast, L-selectin ligand activity of HL60 cells wascompletely eliminated by digestion with OSGE (p<0.001) (Table 3), andsignificantly inhibited following mocarhagin digestion (p<0.001) and bytreatment with functional blocking anti-PSG1-1 PL-1 monoclonal antibody(p<0.002) (Table 3). The effectiveness of OSGE and mocarhagin treatmentswere confirmed by flow cytometric analysis of the sensitive epitopes onCD34 and PSGL-1 with moAb QBEND-10 and moAb PSL-275, respectively.Interestingly, the fact that L-selectin ligand activity on HL60 cellswas completely eliminated following OSGE digestion, but not by PL-1 moAbor mocarhagin treatments, suggests that HL60 cells express othernon-PSGL-1,0-sialoglycoprotein L-selectin ligands. These data areconsistent with previous studies demonstrating the expression ofOSGE-sensitive, non-PSGL-1 L-selectin ligand(s) on HL60 cells (Ramos,C., Smith, M. J., Snapp, K. R., Kansas, G. S., Stickney, G. W., Ley, K.and Lawrence, M. B. (1998) Blood 91(3), 1067-1075).

TABLE 3 L-selectin Ligand Activity of KG1a and HL60 Cells FollowingEnzymatic or Blocking Antibody Cell Treatments under Hydrodynamic ShearFlow¹ % Control Mean Lymphocyte Cells and Treatments Binding² KG1aCells + mocarhagin⁴ 110.0 ± 10.5 + OSGE (60 μg/ml)⁵ 104.2 ± 17.1 + PL-1(anti-PSGL-1; 10 μg/ml) 104.8 ± 18.0 + neuraminidase (Neur.) (0.1 U/ml) 0.3 ± 0.8* + Neur. + DMSO 103.3 ± 12.3 + Neur. + Tunicamycin (15 μg/ml) 34.3 ± 9.8* HL60 Cells + mocarhagin  50.0 ± 2.0* + OSGE  12.5 ± 0.2* +PL-1  62.5 ± 1.0** Negative Controls³  <0.5 ± 0.3* ¹Using theparallel-plate flow chamber, thoracic duct lymphocytes (10 ml/ml H/Hwith Ca⁺⁺) were perfused over glutaraldehyde-fixed monolayers of cellstreated with either mocarhagin (10 μg/ml; 1 hr at 37° C.), OSGE (60μg/ml; 1 hr at 37° C.), PL-1 (10 μg/ml; 30 min. on ice) at a definedshear stress of 4.4 dynes/cm². ²Mean lymphocyte binding from 5 fields ofview from triplicate samples and a minimum of 3 experiments was dividedby the mean lymphocyte binding of the untreated control cells for eachrespective treatment group. ³Negative control groups consisted of 5 mMEDTA containing assay medium and anti-L-selectin antibody-treated(HRL-1; 10 μg/ml) rat lymphocytes. ⁴Mocarhagin digestion was verified bythe inability of anti-PSGL-1 moAb PSL-275 to recognize the P-andL-selectin binding determinant or mocarhagin-sensitive epitope on PSGL-1by flow cytometry. ⁵OSGE activity was confirmed by the inability ofanti-CD34 Qbend-10 to recognize its OSGE-sensitive epitope on CD34 byflow cytometry. *Statistically significant difference in lymphocytyebinding compared with untreated control cells; Student's paired t-test,p < 0.001. **Statistically significant difference in lymphocyte bindingcompared with untreated control cells; Student's paired t-test, p <0.002.

To further investigate the L-selectin ligand activities of HCELL andPSGL-1 expressed on human HCs, Stamper Woodruff assays ofL-selectin-mediated lymphocyte adherence to glutaraldehyde-fixed HCmonolayers under a range of rpms were performed. KG1a cells possessedHCELL ligand activity from >40-120 rpm, which was maximal at 80 rpm,while HL60 cells exhibited L-selectin ligand activity predominantly at80 rpm (FIG. 12C). In addition, L-selectin-mediated lymphocyte adherenceto KG1a cells was 10-fold higher than that of HL60 cells at 80 rpm, andthere was no evidence of lymphocyte binding to K562 and RPMI-8402 cells(FIG. 12C). Since the primary L-selectin ligand on HL60 cells is PSGL-1and its expression is equivalent on KG1a and HL60 cells, these datafurther suggest that, on a per cell basis, KG1a HCELL activity possessesa higher capacity to function as a ligand over a wider range of shearstress.

Though the level of expression of PSGL-1 is equivalent between HL60 andKG1a cells, it was examined whether PSGL-1 on KG1a cells was functioningequivalently to that of PSGL-1 on HL60 cells. Since the criticalN-terminal binding determinant of PSGL-1 for P-selectin overlaps withthe structural binding determinant(s) for L-selectin (Snapp, K. R.,Ding, H., Atkins, K., Warnke, R., Luscinskas, F. W. and Kansas, G. S.(1998) Blood 91, 154-164; De Luca, M., Dunlop, L. C., Andrews, R. K.,Flannery, J. V., Ettling, R., Cumming, D. A., Veldman G. M. and Berndt,M. C. (1995) J. Biol. Chem. 270(45), 26734-26737), it was reasoned thatP-selectin binding capabilities of KG1a and HL60 cells correlates withthe efficiency of PSGL-1 binding to L-selectin. Thus, flow chamberassays of P-selectin ligand activity utilizing Chinese hamster ovarycells transfected with cDNA encoding full-length human P-selectin(CHO-P) was performed. Both HL60 and KG1a cells supported equivalentPSGL-1-mediated CHO-P cell rolling, and K562 and RPMI-8402 cells did notpossess any activity (FIG. 13A). P-selectin ligand activity on KG1a andHL60 cells was prevented following mocarhagin treatment (FIG. 13B).Unlike the differential capability to support L-selectin ligand activitybetween KG1a and HL60 cells, these data suggested that native PSGL-1 asexpressed in the cell membrane was similar both structurally andfunctionally in these cell lines. Of note, RPMI-8402 PSGL-1 wasnon-functional as both an L- or P-selectin ligand, consistent with afinding that PSGL-1 on certain lymphoid cells is non-functional due to alack of activity of α1,3 fucosyltransferases and core 2 β1,6N-acetylglucosaminyltransferases required for creation of a bioactiveligand (Vachino, G, Chang, X.-J., Veldman, G. M., Kumar, R., Sako, D.,Fouser, L. A., Berndt, M. C. and Cumming, D. A. (1995) J. Biol. Chem.270(37), 21966-21974).

C. HCELL is the Preferred L-selectin Ligand on Human HCs.

The distinction in L-selectin ligand activity between HCELL and PSGL-1on whole cells may reflect differences in surface density and/ormembrane topography of the expression of these molecules. To directlycompare the binding capacities of HCELL and PSGL-1, conventional,shear-based Stamper Woodruff assays of L-selectin-mediated lymphocyteadherence to molar equivalents of either HCELL or PSGL-1 immunoaffinitypurified from KG1a and HL60 cells, and from a de novo AML (M1) wasperformed.

To isolate HCELL and PSGL-1 for cell binding experiments, KG1a CD44(Hermes-1 rat IgG) and PSGL-1 (PL-2 mouse IgG) from cell membraneprotein preparations were immunoaffinity purified. Autoradiography ofHermes-1 and PL-2 immunoprecipitates obtained from whole cell lysates of[³⁵S]-metabolically radiolabeled KG1a cells showed the specificity ofHermes 1 and PL-2 for their respective antigens (FIG. 14A), revealingthat the 100 kDa form of CD44 was principally isolated and that bothdimer (˜220 kDa) and monomer (˜140 kDa) isoforms of PSGL-1 were isolated(FIG. 14A). There was a minor contaminant protein of 30 kDaimmunoprecipitated by Hermes 1, which was removed by subsequent passageof immunoprecipitates through a 50 kDa MW cut-off filter. To normalizefor molar equivalency of purified protein utilized in Stamper-Woodruffassays, the densitometric optical density (OD) of[³⁵S]-methionine-labeled CD44 and PSGL-1 (each passed through 50 kDacut-off filter) on autoradiograms of immunoaffinity purified materialspotted onto glass was compared. It was found that the OD of 1 μg PSGL-1was 2-fold greater than the OD of 0.75 μg CD44. Since monomer PSGL-1(140 kDa) is ˜1.4-fold higher MW than CD44 (100 kDa) and since PSGL-1has twice as many methionine residues than CD44 (13 vs. 6), the two-foldhigher signal of PSGL-1 indicated that 0.75 μg CD44 and 1 μg PSGL-1represent equimolar amounts of the respective proteins. Using theseequimolar amounts, CD44 supported up to 10-fold greater lymphocytebinding compared with PSGL-1 at 80 rpm, and supported a 5- to 10-foldhigher lymphocyte adherence compared with PSGL-1 from 40 to 100 rpm(FIG. 14B). CD34 and L-selectin immunoprecipitated from KG1a cells(negative molecular controls) did not support any L-selectin-mediatedlymphocyte adherence (FIG. 14B). These data directly comparing therelative L-selectin binding efficiencies of purified CD44 and PSGL-1were similar to results obtained from whole cell analysis.

To explore whether disparate HCELL and PSGL-1 L-selectin ligandactivities were also present in native human hematopoietic cells, theL-selectin ligand activity of HCELL and PSGL-1 on circulating blastsfrom a patient with an AML without maturation (M1) was investigated. Inpreliminary Stamper-Woodruff assays of whole AML (M1) cell activities,AML (M1) cells showed comparable L-selectin ligand activity to that ofKG1a cells, and the expression of CD44 and PSGL-1 (measured by flowcytometry) was also similar to that of KG1a. CD44 and PSGL-1 from thesecells was immunoprecipitated and their binding capacity byStamper-Woodruff assay (80 rpm) was examined. In parallel, L-selectinligand activities of HCELL and PSGL-1 from KG1a and HL60 cells were alsoassessed, and digestions with N-glycosidase-F and OSGE were performedfor comparative analysis. CD44 from both KG1a and AML (M1) supportedsignificantly greater L-selectin-mediated lymphocyte adherence thanPSGL-1 from KG1a, HL60 or AML (M1) (3-fold higher mean number oflymphocytes bound to CD44 than to PSGL-1; p<0.001) (Table 4). Inaddition, neither CD44 from HL60, isotype control immunoprecipitates,CD34 and L-selectin immunoprecipitates, or neuraminidase-treated CD44and PSGL-1 immunoprecipitates from KG1a cells (Table 4) possessed anyL-selectin ligand activity. Similar to N-glycosidase-F treated CD44 fromKG1a cells, the L-selectin binding activity of N-glycosidase-F treatedAML M1 CD44 was markedly reduced (to background levels) (Table 4).Interestingly, PSGL-1 isolated from KG1a cells and AML (M1) blastspossessed a greater capacity to engage with L-selectin than PSGL-1 fromHL60 cells (Table 4), even though P-selectin mediated binding to nativePSGL-1 was identical between KG1a and HL60 cells (FIGS. 13A and 13B).Photomicrographs of lymphocytes bound to CD44 or PSGL-1 inStamper-Woodruff assays illustrated the distinctive differences in therange of L-selectin ligand activity of KG1a CD44 and AML (M1) CD44(FIGS. 15A and 15D, respectively) compared to KG1a PSGL-1 (FIG. 15G);even at 3-fold molar excess of KG1a PSGL-1 (FIG. 15H), L-selectin ligandactivity of CD44 (FIG. 15A) was still greater than that of PSGL-1.N-glycosidase-F (FIGS. 15B and 15E) and OSGE (FIG. 15I) treatmentsmarkedly diminished lymphocyte binding comparable to isotype controllevels (FIGS. 15C and 15F) confirming the relevant contributions ofN-glycans and O-glycans on HCELL and PSGL-1, respectively.

TABLE 4 L-selectin Ligand Activity of Immunoprecipitated CD44 and PSGL-1from KG1a, HL60 Cells and Blasts from an AML (M1) in theStamper-Woodruff Assay¹ Cellular L-selectin Ligand Mean Number of BoundLymphocytes² KG1a CD44 357.8 ± 36.6  N-glycosidase-F CD44  30.7 ± 18.4*Isotype Control 23.3 ± 7.1* PSGL-1  44.8 ± 6.7** Isotype Control 11.0 ±1.6* CD34 (Molecular Control)  4.0 ± 2.0** L-selectin (MolecularControl)  0.5 ± 0.6** Isotype Control 8.5 ± 3.5 HL60 CD44 6.5 ± 3.6Isotype Control 5.4 ± 2.3 PSGL-1 11.0 ± 2.1* Isotype Control  3.1 ± 2.9*AML (M1) CD44 425.3 ± 9.5  N-glycosidase-F CD44 23.4 ± 6.1* IsotypeControl 34.3 ± 4.1* PSGL-1  141.5 ± 22.6** Isotype Control 19.5 ± 6.2*¹Rat thoracic duct lymphocytes (1 × 10⁷/ml) were overlayed ontoglutaraldehyde-fixed spots of immunoaffinity purified CD44 (Hermes-1),N-glycosidase-F-treated CD44, CD34 (QBend-10), L-selectin (LAM 1-3) orisotype control (rat IgG or mouse IgG) (each at 0.75 μg/spot), or PSGL-1(PL-2) (1 μg/spot) and incubated on a shaker at 80 rpm at 4° C. ²Theaverage number of bound lymphocytes from 5 fields of view at 100X mag.from duplicate slides and a minimum of three experiments. Eachexperiment included anti-L-selectin Ab-treated (HRL-1; 10 μg/ml) orPMA-treated lymphocytes and 5 mM EDTA containing assay medium groups, orVibrio cholerae neuraminidase-treated spots (ligand), which allcompletely eliminated lymphocyte binding (<0.8 mean number of boundlymphocytes). *Statistically significant difference in lymphocytebinding to treatment or isotype immunoprecipitate compared with eachrespective untreated cell molecule; Student's t-test, p < 0.001.**Statistically significant difference in lymphocyte binding to thesegroups compared with lymphocyte binding to untreated KG1a and AML (M1)CD44; Student's t-test, p < 0.001.

To gain insight into the higher capacity of HCELL to bind to L-selectincompared with that of PSGL-1, the expression of the sialyl-fucosylatedstructures (recognized by rat moAb HECA-452) on CD44 and on PSGL-1 wasexamined, which correlates with L-selectin binding capacity. SDS-PAGEand HECA-452 immunoblot analysis of equimolar amounts of CD44 and PSGL-1immunoprecipitated from KG1a membrane proteins revealed that CD44 wasdistinctly more HECA-452-reactive than PSGL-1 (FIG. 16), suggesting thatHCELL contains a greater number of HECA-452 epitope(s) than PSGL-1,which could account for its higher avidity towards L-selectin.

Example 11 Immunoaffinity Purification of HCELL and PSGL-1

HCELL and PSGL-1 were immunoprecipitated from human HCs. Theimmunoprecipitation procedure was followed as described (Dimitroff, C.J., Lee, J. Y., Fuhlbrigge, R. C. and Sackstein, R. (2000) Proc. Natl.Acad. Sci. 97(25), 13841-13846). Briefly, membrane proteins of human HCs(Dimitroff, C. J., Lee, J. Y., Fuhlbrigge, R. C. and Sackstein, R.(2000) Proc. Natl. Acad. Sci. 97(25), 13841-13846) were firstsolubilized in 2% NP-40 and precleared in Protein G-agarose (LifeTechnologies). Membrane protein was then quantified using Bradfordprotein assay (Sigma). Solubilized and precleared membrane proteinpreparations (including [³⁵S]-methionine-radiolabeled membrane proteinsand whole cell lysates (Dimitroff, C. J., Lee, J. Y., Fuhlbrigge, R. C.and Sackstein, R. (2000) Proc. Natl. Acad. Sci. 97(25), 13841-13846))were then incubated with anti-PSGL-1 Ab PL-2 or anti-CD44 monoclonal AbHermes-1 (a ratio of 100 μg protein:3 μg antibody) for 18 hours at 4° C.on a rotator. Immunoprecipitations with anti-CD34 antibody QBEND-10,anti-L-selectin antibody (LAM1-3) or mouse IgG/rat IgG isotype controlswere also performed to serve as negative controls. The antibody-lysatemixture was added to Protein G-Agarose and incubated for 1 hr at 4° C.under constant rotation. For SDS-PAGE/Western blotting,immunoprecipitates were washed 5-times with lysis buffer/2% NP-40/1%SDS/1% BSA and 3-times lysis buffer without BSA, and boiled in reducingsample buffer for analysis. For functional assessment of CD44 or PSGL-1in adherence assays, respective immunoprecipitates were washed 5-timeswith lysis buffer/2% NP-40/1% SDS/1% BSA and 3-times lysis bufferwithout NP-40, SDS or BSA, then suspended in PBS and boiled for 5 min.to dissociate CD44 or PSGL-1 from immune complexes. Eluates were thenpassed through 50 kDa MW cut-off Microcon® filters (Fisher Scientific,Inc.). Protein concentration of the retentate was measured by Bradfordprotein determination assay.

Parallel experiments were performed wherein respective antibodies wereincubated with Protein G-agarose, and immunoprecipitates were boiled inPBS as mentioned above. Eluates analyzed by SDS-PAGE and Coomassie bluestaining revealed that >99% of the antibody was still bound to ProteinG-agarose after boiling in PBS and, therefore, contributed negligibly tothe protein levels quantified. Moreover, with exception of a minorcontaminating band at 30 kDa in immunoprecipitates of Hermes-1, othernon-CD44 and non-PSGL-1 proteins, respectively, were not isolated byimmunoprecipation using Hermes-1 and PL-2 (see autoradiograms ofimmunoprecipitates obtained from whole cell lysates (7) of[³⁵S]-metabolically radiolabeled KG1a cells, FIG. 14A). The use of the50 kDa cut-off filter removed the contaminating protein of 30 kDaimmunoprecipitated by Hermes-1, yielding pure KG1a CD44.

To confirm the contribution of N-glycans on HCELL or O-sialoglycans onPSGL-1, membrane preparations were first treated with eitherN-glycosidase-F (0.8 U/ml) or OSGE prior to immunoprecipitation asdescribed (Dimitroff, C. J., Lee, J. Y., Fuhlbrigge, R. C. andSackstein, R. (2000) Proc. Natl. Acad. Sci. 97(25), 13841-13846).Immunoaffinity purified material (0.75-3 μg/spot) was spotted onto glassslides for analyses in the Stamper-Woodruff assay (See below for assaydescription). For autoradiographic analysis of immunoprecipitated[³⁵S]-methionine-radiolabeled CD44 and PSGL-1, immunoprecipitates werespotted onto glass slides, allowed to dry and exposed Kodak BioMax-MRfilm for 12 h at −80° C. Densitometric analysis (optical density) of theautoradiograms was performed with a Hewlett-Packard Scanj et 5200Cscanner and NIH Image processing and analysis program.

Example 12 RT-PCR Analysis of α1,3 Fucosyltransferases (FucTIV andFucTVII) and α2,3 Sialyltransferase (ST3Gal IV)

As HECA-452 expression is dependent on critical sialofucosylations oncore poly-N-acetyllactosaminyl chains, it was investigated whether therelative difference in HECA-452 epitope expression and L-selectinbinding activity was a consequence of up-regulated α2,3sialyltransferase (ST3Gal IV) and leukocyte α1,3 fucosyltransferases(FucTIV and FucTVII), which are required for biosynthesis of HECA-452epitope (Sasaki, K., Watanabe, E., Kawashima, K., Sekine, S., Dohi, T.,Oshima, M., Hanai, N., Nishi, T. and Hasegawa., M. (1993) J. Biol. Chem.268(30), 22782-22787: Fuhibrigge, R. C., Kieffer, D., Armerding, D. andKupper, T. S. (1997) Nature 389, 978-981; Zollner, O. and Vestweber, D.M. (1996) J. Biol. Chem. 271, 33002-33008; Goelz, S., Kumar, R., Potvin,B., Sundaram, S., Brickelmaier, M. and Stanley, P. (1994) J. Biol. Chem.269, 1033-1040).

Total cellular RNA was extracted with Trizol® LS reagent according tomanufacturer's protocol (Gibco, Life Sciences) and utilized in theTitan™ One Tube RT-PCR System (Roche Molecular Biochemicals). ST3Gal IVsense 5′-ctctccgatatctgttttattttcccateccagagagaagaaggag-3′ andanti-sense 5′-gattaaggtaccaggtcagaaggacgtgaggttctt-3′ primers andthermal cycling conditions [RT at 52° C. for 45 minutes, 1 cycle at 94°C. for 2 minutes, 30 cycles at 94° C. for 1 minute, 57° C. for 1 minuteand 72° C. for 2 minutes, and 1 cycle at 68° C. for 7 minutes] were usedto amplify a 0.96 kb cDNA fragment of ST3Gal IV. To amplify a 0.55 kbcDNA fragment of FucTVII (GenBank, Accn#U08112), specific primers, sense5′-cccaccgtggcccagtaccgcttct-3′ and anti-sense5′-ctgacctctgtgcccagcctcccgt-3′ and thermal cycling conditions [RT at52° C. for 45 minutes, 1 cycle at 94° C. for 2 minutes, 30 cycles at 94°C. for 30 seconds, 60° C. for 45 seconds and 72° C. for 1 minute, and 1cycle at 68° C. for 7 minutes] were used. Using identical RT and thermalcycling conditions, a 0.50 kb cDNA fragment of FucTIV was generated fromsense 5′-cgggtgtgccaggctgtacagagg-3′ and anti-sense5′-tcgggaacagttgtgtatgagatt-3′ primers (GenBank, Accn#M58597).

RT-PCR analysis of FucTIV and FucTVII and of ST3Gal IV gene expressionin KG1a, HL60, K562 and RPMI-8402 cells showed that FucT IV expressionwas relatively similar in all cell lines (FIGS. 17A and 17B), but theFucTVII expression was highest in HL60 and KG1a cells (Lane 1, FucTIV,and Lane 2, FucTVII; FIG. 17A). Interestingly, ST3Gal IV (Lane 1; FIG.6B) was expressed at a high level in KG1a cells and at a very low levelin all other cell lines, suggesting that the inherent level of ST3Gal IVmay help regulate the expression of relevant HECA-452-reactivestructures and critical L-selectin binding determinants on CD44 and/orPSGL-1.

HECA-452 expression is dependent on critical sialofucosylations on corepoly-N-acetyllactosaminyl chains. the relative difference in HECA-452epitope expression and L-selectin binding activity was a consequence ofup-regulated α2,3 sialyltransferase (ST3Gal IV) and leukocyte α1,3fucosyltransferases (FucTIV and FucTVII), which are required forbiosynthesis of HECA-452 epitope was investigated. RT-PCR analysis ofFucTIV and FucTVII and of ST3Gal IV gene expression in KG1a, HL60, K562and RPMI-8402 cells showed that FucT IV expression was relativelysimilar in all cell lines (FIGS. 17A and 17B), but the FucTVIIexpression was highest in HL60 and KG1a cells (Lane 1, FucTIV, and Lane2, FucTVII; FIG. 17A). Interestingly, ST3Gal IV (Lane 1; FIG. 17B) wasexpressed at a high level in KG1a cells and at a very low level in allother cell lines, suggesting that the inherent level of ST3Gal IV mayhelp regulate the expression of relevant HECA-452-reactive structuresand critical L-selectin binding determinants on KG1a CD44 and/or PSGL-1.

EQUIVALENTS

From the foregoing detailed description of the specific embodiments ofthe invention, it should be apparent that novel compositions andapplications have been described. Although particular embodiments havebeen disclosed herein in detail, this has been done by way of examplefor purposes of illustration only, and is not intended to be limitingwith respect to the scope of the appended claims which follow. Inparticular, it is contemplated by the inventor that varioussubstitutions, alterations, and modifications may be made to theinvention without departing from the spirit and scope of the inventionas defined by the claims.

1. An composition comprising a population of cells enriched from blood,said cells expressing a glycosylated CD44 polypeptide, wherein saidglycosylated CD44 polypeptide comprises sialylated, fucosylated glycans,and wherein said glycosylated CD44 polypeptide is a ligand forE-selectin, L-selectin, or both.
 2. The composition of claim 1, whereinthe CD44 polypeptide comprises the amino acid sequence of SEQ ID NO: 1.3. The composition of claim 1, wherein the cells are stem cells.
 4. Amethod of treating a hematopoietic disorder in a mammal, the methodcomprising administering to said mammal a composition comprising thecells of claim
 1. 5. A method of treating cancer in a mammal, the methodcomprising administering to said mammal a composition comprising thecells of claim 1.